RIBAT NATIONAL UNIVERSITY
INSTITUTE OF FORENSIC SCIENCES
TheToxicological Effects of Paraphenylenediamine on
Albino Rats
A thesis submitted for the partial fulfillment of M.Sc. degree in forensic
science
Submitted by:-
Elkhomainy Mohamed Abuelgasim Nasir
Supervisor:-
Dr.Amir Sadig Mahmud
2017
بسم اهلل الرحمن الرحيم
ل هل جزاء الحسان إ ق ال تعالي )
(06)الحسان
(06)فبأي آلء ربكما تكذبان (06(,)06اآليات ) الرحمن,
صدق اهلل العظيم
Dedication
I dedicate this research for my mother, who taught me that even the largest task can be accomplished if it is done one step
at a time.
It is also dedicated this thesis to my father, who taught me that the best kind of knowledge to have is that which is
learned for its own sake.
To my best friends, Mohamed ALamin Allaithy and Hatim Bakri Allaithy who are contributes in my successes.
To You all.
Acknowledgements
I would like to deliver my thanks and gratitude to Dr.
Amir Sadig Mahmoud,
who has been the ideal thesis supervisor. for His sage
advice, insightful criticisms, and patient encouragement
aided the writing of this thesis in innumerable waves.
Also I would like to thanks dr. Ahmed Awad Algamel, the
director of forensic science institute, for his supporting,
advice and continuous learning.
Also thanks extended to my college Rumaisa Bushra Idrees
for her great efforts and helpful.
And thanks for all my friends.
Abstract
This study was done on 40 Albino-type rats to investigate the possible toxic effect
of the hair dye doses.
The dye was dissolved in distilled water in concentrations of 10 mg, 20 mg, 30 mg
and given orally to Albino rats.
The first group called control group.the second group was given (10 mg/kg b.w).
the third group was given (20 mg/kg b.w). the fourth group was given (30 mg/kg
b.w) of hair dye.
The effect of hair dye result in some clinical symptoms in all groups except control
group. Such as neck and face odema, general weakness and tremors.
Serobiochemical changes were increases in the activities of GOT (AST), GPT
(ALT) and decreases in total protein, albumin for liver function.
Also increases in level of urea and creatinine for renal function. and decreases in
cholesterol level.
Haematological changes were decreases in haemoglobin, red blood cells count,
PCV, MCV and MCH. And increases MCHC values in all groups except control
group.
الخـــالصة
علي عدد أربعين من الفئران من ساللة )ألبينو( وذلك لدراسة التأثير أجريت هذه التجربة -
السمي للجرعات المختلفة لصبغة الشعر.
قسمت الفئران إلي أربعة مجموعات متساوية وقد غذيت جميع المجموعات علي نوع واحد -
أعطيت من الغذاء في البداية. المجموعة األولي سميت المجموعة الضابطة. المجموعة الثانية
((10mg/kg b.w من صبغة الشعر لكل فأر من المجموعة. المجموعة الثالثة أعطيت
((20mg/kg b.w من صبغة الشعر لكل فأر من المجموعة. أما فئران المجموعة الرابعة
من صبغة الشعر لكل فأر من المجموعة. 30mg/kg b.w)فقد أعطيت )
ض السريرية في كل المجموعات عدا تسبب التأثير بصبغة الشعر بظهور بعض األعرا -
المجموعة الضابطة مثل تورم العنق والوجه والضعف العام وبعض اإلرتعاشات.
عند إجراء اإلختبارات البيوكيميائية لمعرفة األثر السمي علي الكبد كان هنالك إرتفاع في -
تفاع في , البروتين الكلي, األلبيومين وأيضا إرGPTوالـ GOTمعدالت إنزيمات الكبد
معدالت اليوريا والكرياتينين فيما يختص بوظائف الكلي. أيضا هنالك نقصان في مستوي
الضابطة, عند فحص الدم كان هنالك المجموعة الكوليستيرول في كل المجموعات عدا
وذيادة في RBCs count, PCV, MCV, MCHإنخفاض في معدالت الهيموقلوبين,
. MCHCمعدالت الـ
Table of contents
Issue Page
Dedication І
Acknowledgment І І
Abstract English І І І
Abstract Arabic ІV
Table of Contents V
List of Tables VІІ
List of Figures VІІІ
List of Abbreviations ІX
Appendix XІ
1.Chapter One: Introduction and Literature Review
1.1 Introduction 1
1.2 Previous studies 6
1.3 Uses of PPD 9
1.4 Acute toxicity 9
1.5 Treatment of oral toxicity 10
1.6 Investigation of PPD 11
1.7 Safety 11
1.8 Mechanism of action 12
1.9 Patch tests 13
1.10 Objective 14
2. Chapter Tow: Materials & Methodology
2.1 Methodology 15
2.1.1 Clinical chemistry 16
2.1.2 Methods used for determination of serum constituents 16
2.1.3 Liver function tests 16
2.1.3.1 Glutamic oxaloacetic transaminase (GOT/AST) 16
2.1.3.2 Glutamic pyruvic transaminase (GPT/ALT) 18
2.1.3.3 Total protein 19
2.1.3.4 Albumin 20
2.1.3.5 Cholesterol 21
2.1.4 Renal function tests 22
2.1.4.1 Urea Estimation 22
2.1.4.2 Creatinine Estimation 23
2.2 Haematology 24
2.2.1 Haematological methods 24
2.2.2 Haemoglobin concentration (HB) 24
2.2.3 Packed cell volume (PCV) 25
2.2.4 Red blood cell (RBC) count 25
2.2.5 Mean corpuscular volume (MCV) 25
2.2.6 Mean corpuscular haemoglobin concentration (MCHC) 26
2.2.7 Mean corpuscular haemoglobin(MCH) 26
2.3 Statistical methods 27
3. Chapter Three: Results
3.1 Results 28
4. Chapter Four: Discussion & Recommendations
4.1 Discussion 38
4.2 Recommendations 40
References 41
List of tables
No Table title Page No.
Table (3.1) Biochemical parameters of serum samples 28
Table (3.2) Haematological parameters 32
Table (3.3) Biochemical parameters 36
List of figures
No Figure title Page
Figure (3.1) Showing (GOT) concentration differences 29
Figure (3.2) Showing (GPT) concentration differences 29
Figure (3.3) Showing (T.protein) concentration differences 30
Figure (3.4) Showing (Cholesterol) concentration differences 30
Figure (3.5) Showing (Albumin) concentration differences 31
Figure (3.6) Showing (PCV) concentration differences 33
Figure (3.7) Showing (HB) concentration differences 33
Figure (3.8) Showing (RBCs) concentration differences 34
Figure (3.9) Showing (MCH) concentration differences 34
Figure (3.10) Showing (MCV) concentration differences 35
Figure (3.11) Showing (MCHC) concentration differences 35
Figure (3.12) Showing (Urea concentration) differences 37
Figure (3.13) Showing (Creatinine concetration) differences 37
List of abbreviations
Abbreviations Stands for:-
PPD ParaPhenyleneDiamine
ARF Acute Renal Failure
AKI Acute Kidney Injury
ICP/MS Inducted Coupled Plasma / Mass Spectrometry
HPLC/MS High Performance Liquid Chromatography / Mass Spectrometry
GC/MS Gas Chromatography / Mass Spectrometry
SIM MODE Selective Ion Monitoring .. Gas Chromatography / Mass Spectrometry
PAP Paraamenophenol
AA Arylamines
LD50 Lethal dose which kill half of population
MALDI-
MS/MS
Matrix-Assisted-Laser-Desorption/Ionization
Mass Spectrometry
MAPPD Mono Acetylated - paraphenylene diamine
DAPPD Di Acetylated - paraphenylene diamine
EDTA Ethylene Diamine Tetra Acetic acid
RPM Round Per Minute
GOT Glutamic Oxaloacetic Transaminase
GPT Glutamuc Pyruvic Transaminase
HB Haemoglobin %
PCV Packed Cell Volume
RBCs Red Blood Cells
MCV Mean Corpuscular Volume
MCHC Mean Corpuscular Haemoglobin Concentration
MCH Mean Corpuscular Haemoglobin
AST Aspartate Transaminase
ALT Alanine Transaminase
SPSS Statistical Package for Social Sciences
Chapter One
Introduction and Literature Review
Chapter Tow
Materials and Methodology
Chapter Three
Results
Chapter Four
Discussions & Recommendations
Appendix
- AST Measurement:-
R1= GOT substrate (Aspartate 100 mmol/L,Ketoglutarate 2 mmol/L).
R2= DNPH (2,4-dinitrophenylhidrazaine) 1 mmol/L.
R3= NaOH 0.4 N.
- ALT Measurement:-
R1= GPT substrate (Alanine 200 mmol/L,Ketoglutarate 2 mmol/L).
R2= DNPH (2,4-dinitrophenylhidrazaine) 1 mmol/L.
R3= NaOH 0.4 N.
- Determination of Total protein:-
R1= Biuret reagent (Cupric sulfate 6 mmol/L,sodium-potassium-tartrate 21
mmol/L,potassium iodide 6 mmol/L,sodium hydroxide 0.75 mmol/L).
Total protein standard concentration= 7 g/dl.
- Determination of Albumin:-
R1= (BCG) Bromocresol reagent (Succinate buffer 75 mmol/L, PH 4.2, BCG
0.12 mmol/L, tensioactive 2g/L).
Albumin standard concentration= 5 g/dl.
- Cholesterol measurement:-
R1= Monoreagent (PIPES 200 mmol/L, pH 7.0, sodium cholate 1 mmol/L,
cholesterol esterase > 250 UL, cholesterol oxidase > 250 U/L, peroxidase > 1
KU/L, 4-aminoantipyrine 0.33 mmol/L, ADPS 0.4 mmol/L, non-ionic tensioactive
2 g/l).
Cholesterol standard concentration= 200 mg/dl.
- Urea Estimation:-
R = sodium salicylate 62 mmol/L, sodium nitroprusside 3.4 mmol/L, phosphate
buffer 20 mmol/L, pH 6.9.
- Creatinine Estimation:-
R = sodium hydroxide 0.4 mol/L detergent.
- Haemoglobin concentration:-
Drabkin`s solution = 0.2g Potassium cyanide, 0.2g potassium ferriccyanide, and
1g sodiuh bicarbonate per 1 litter of distilled water.
1. Introduction & Literature Review:-
1.1. Introduction:-
Paraphenylenediamine (PPD), a derivative of paranitroaniline, has been used
fordyeing furs, photochemical measurements, accelerating vulcanization and azo-
dye manufacturing, as well as for oxidizing hair dyes. Chemically, it is an aromatic
diamine related to aniline, both accidental and intentional ingestion of PPD is
frequently reported from Africa, the Middle-East, and the Indian subcontinent (1) (2)
(3) where PPD is commonly mixed with henna, which is traditionally applied to color
the palms of hands and to dye the hairs. PPD accelerates the dyeing process.
The toxicity of PPD includes skin irritation, contact dermatitis, chemosis,
lacrimation, exophthalmos, or even permanent blindness, due to local contact.
Ingestion of PPD produces two types of toxic effects. The first consists of rapid
development of severe oedema of the face, neck, pharynx, tongue, and larynx with
respiratory distress, often requiring tracheostomy, in the later phase, rhabdomyolysis
and acute tubular necrosis supervene (4).
Vomiting, gastritis, hypertension, vertigo, tremors, and convulsions have been
reported (5).
suicide is a preventable public health problem, resulting in one million fatalities
every year worldwide, increasing by 60% over the last 50 years especially in
developing countries(6) Poisoning is a preferred method of suicide and is one of the
major problems encountered in emergency departments of hospitals,(7)
Poisoning
with hair dye containing paraphenylenediamine is a new trend of intentional self
harm in various developing countries of Asia and Africa (8) and is associated with
high mortality. (9)
PPD is an active ingredient of ‘Kala Pathar’. It is crushed and mixed with henna and
used as hair dye for enhancing its color (10)
.
PPD ingestion causes symptoms involving different organs. Chemically, it is a
derivative of paraphenylaniline, brown or black color solid substance, easily soluble
in hydrogen peroxide and not in water. It is a good hydrogen donor and metabolized
by electron oxidation to an active radical by cytochrome P450 peroxidase to form a
reactive compound called benzoquinone diamine. This can be further oxidized to a
trimer known as Brandowaski's base, a well known compound, reported to cause
anaphylaxis and mutation. (10)
Ingestion of PPD causes rapid development of edema of the face, neck, pharynx,
tongue and larynx initially and rhabdomyolysis followed by acute renal failure
(ARF) as renal tubular necrosis occurs due to the deposits of the toxic metabolites
of PPD. (11)
The compound PPD is highly toxic. When taken orally death occurs within the first
6-24 hours due to angioneurotic edema(10) Smaller doses cause angioneurotic edema
and hepatitis while moderate doses cause acute renal failure within the first week.
As there has been a recent increase in frequency of hair dye poisoning , this study
was done to study clinical profile of Paraphenylenediamine containing hair dye
poisoning and outcome among experimental animals (rats).
Hair dye present as a white solid to red crystalline powder but samples can darken
due to air oxidation. (12)
The main component of hair dye causing toxicity is ParaPhenylenediamine (PPD)
which is an organic compound with the formula C6H4 (NH2)2. This derivative of
aniline.
The PPD is an aromatic amine not found in nature and many industrial companies
produce it commercially.
Its used in the manufacture of hair and other dyes , syntactic fibers, and polyurethane,
as an additive to gasoline, in photographic developing and in intermediate in the
manufacture of rubber and antioxidants.
Acute poisoning by PPD causes characteristic severe angioedema of the upper
airway accompanied by a swollen, dry, hard, and protruding tongue.
Systemic intoxication results in multisystem involvement and can cause
rhabdomyolysis, acute renal failure (ARF), PPD consumption is an uncommon cause
of ARF, also can cause vertigo, tremor, convulsions and sometimes coma. There is
no specific antidote for PPD and treatment is mainly supportive.(13)
The extent of renal involvement in poisoning varies between transient proteinuria
and oliguric acute kidney injury (AKI), AKI commonly develops a few days after
PPD exposure. The mechanisms of kidney injury following hair dye poisoning are
many.
The PPD itself has a direct toxic effect on kidney due to its aromatic structure, which
makes its easy reabsorption and concentration in tubule and can cause ARF. (13)
It can cause rhabdomyolysis with the deposition of myoglobin cast within the renal
tubules and hemolysis with resultant hemoglobinuria causing acute tubular necrosis
and ARF. The hypovolemia in addition can complicate renal failure.
Stone hair dye is a commercial hair coloring. It contains PPD and other elements
(14)(15)(16) Many cases of toxicity and mortality either due to its accidental or deliberate
ingestion were reported in Egypt, Sudan, Israel, Morocco, Saudi Arabia, India and
Tunisia (17) On the other hand, paraphenylenediamine is a very common allergen in
man. Contact dermatitis following exposure to chemical allergens is a common
health problem (18) , it is an immunologic skin reaction that occurs in a genetically
predisposed individual, the risk of sensitization rises with frequency of contacts with
the allergen. Cases of photosensitization induced by paraphenylenediamine have
also been recorded. The hypersensitivity of its para-group (19) N-Substitution of PPD
influences its sensitization potential. Also the length of the chain of the alkyl
substituent often has an effect on the sensitization potential (20)
A number of analytical procedures have been developed to separate and determine
PPD intermediate in coloring products, including ICP/MS, HPLC/MS and GC- MS
(21)(22) Some of these methods require laborious and time consuming methods of
extractions of hair dye components, followed by their chemical derivatization (23)
High performance liquid chromatography (HPLC) has been adopted as a popular
method for the determination PPD derivatives, during which test chemicals are not
heated above room temperature. Each PPD derivative has a characteristic ultraviolet
absorption spectrum, and qualitative analysis is possible by selection of a suitable
detection wavelength. Therefore HPLC is a suitable method for the determination of
PPD derivatives (24) HPLC/MS is a suitable for the determination of PPD derivatives
because of the stability of aromatic imines (25) , PPD was converted into an imine;
taking in account that coloring product composition should not affect the reaction of
On the application of GC/MS technique operating in SIM mode to determine the
derivatives PPD obtained from commercially available hair dyes, many peaks were
obtained in the chromatograms with the use of flame ionization detector. This could
be due to the thermo labile nature of PPD. This explains the previous use of GC/MS
for the qualitative analysis of its derivative (26).
Identify the suitable methods of detection, and quantify its components that may be
potentially toxic on oral or dermal exposure.
The chemical is absorbed through the skin. The type and severity of symptoms vary
depending on the amount of chemical involved and the nature of exposure. PPD is
used in almost every hair color dyes on the market, regardless of brand. The darker
the color, usually the higher the concentration. Even the so-called “natural” and
“herbal” hair colors, while ammonia free, contain PPD (27).
Paraphenylenediamine (PPD) and paraaminophenol (PAP) are examples of
arylamines (AA). When used as hair dyes, these primary intermediates diffuse
readily into the hair shaft where they undergo oxidative chemical reactions or
coupled with other dye precursors (typically met-compounds or hydroxybenzenes)
to form larger molecular weight, colored species which subsequently become
entrapped in the hair shaft, conferring performance to the coloring process. (28).
1.2. Previous studies:-
Study under name of some toxicological health hazards associated with sub-
chronic dermal exposure to paraphenylene-diamine (PPD): An experimental
study said Paraphenylene-diamine (PPD) is a widely used chemical in almost all
hair dye formulations.
The main purpose of using PPD as hair dye ingredients is to fasten the process of
dyeing as compared to traditional henna. Since mammalian contact with these
chemical via the scalp for cosmetic applications such as hair dyes. The current study
was designed to investigate potency of PPD to induce toxic effects upon the liver,
kidney, heart and pancreas after 30 days of continuous daily dermal application in
three different dosages 1, 2, 3 mg/kg in 30 adult male Albino rats, 10 per each group.
Another 10 animals were served as a control group received only distilled water.
PPD was dissolved in doubled distilled water. The rats were painted on their dorsal
side clipped free of fur with PPD solution or vehicle alone. At the end of period of
experiment, rats were scarified by cervical dislocation; blood was collected for
evaluation of hepatic, renal, cardiac functions and the serum glucose level, while
internal organs (liver, kidney) were collected for histopathological examinations.
The results proved that sub chronic dermal exposure to PPD can induce
hyperglycemia, disturbed hepatic, renal and cardiac functions.The histopathological
findings showed that PPD cause mild, moderate, and severe chronic inflammation
in the heart and liver. In the kidney and pancreas it causes moderate and severe
chronic inflammation. In a conclusion, this study established the multivisceral toxic
effects of sub chronic dermal exposure to paraphenylenediamine.
Other study under the name of Paraphenylenediamine poisoning
Said the commonest constituent of all hair dyes is paraphenylenediamine (PPD).
Hair dye poisoning is emerging as one of the causes of intentional self-poisoning to
commit suicide. In this article, we report a case of PPD poisoning and the importance
of clinical of hair dye poisoning. The lack of specific diagnostic tests, a specific
antidote for paraphenylene diamine poisoning and the importance of early
supportive treatment modalities are also discussed.
Also there is study under name Neuro and Nephro-Toxicity in Rats Topically
Treated with Para-PhenyleneDiamine said Para-phenylene diamine ( PPD )
systemic intoxication in Saudi Arabia has been increased over the last decade.
The aim of the study was to provide more insight into PPD intoxication with
reviewing possible underlying mechanisms. The topical treatment with PPD
(2mg/kg) for 5 weeks and its subsequent withdrawal caused decreased in
monoamines (noerepinephrine, dopamine, serotonin and histamine) content in all
brain region (cerebellum, brain stem, striatum, cerebral cortex, hypothalamus and
hippocampus) at different time intervals. Moreover, the present results indicated that
treatment with PPD for 5 weeks and its subsequent withdrawal caused increased in
serum urea and serum creatinine levels. In conclusion. PPD causes serious
multisystem toxicity and its selling to public should be officially restricted to reduce
poisoning by this agent.
Also study under the name of Toxicity Effects of Hair Dye Application on Liver
Function in Experimental Animals said This study was conducted to assess the
hair dye toxicity by using hair dye among experimental rats in order to verify the
biochemical and haematological abnormalities and liver dysfunction.
Methods: Albino Wistar Rats were obtained from the Faculty of Pharmacy,
University of Khartoum– Sudan. The rats were divided into two batches on the basis
of using the commercial hair dye as oral or subcutaneous administration
respectively; each batch has four groups (control and three test groups) each
comprising six rats.
Batch-1 (group-2, 3, and 4 orally administered with 10, 20, and 30mg/kg body
weight of the commercial hair dye, respectively); and Batch-2 (group-2, 3, and 4
subcutaneously administered with 10, 20, and 30 mg/kg body weight of the
commercial hair dye, respectively).
Results: The clinical features were shown in all rats batches, administered orally or
subcutaneously with the commercial hair dye. These clinical features rates from
slight weakness in group 2 to head, neck, and pharyngeal oedema in group-3 up to
severe weakness in hinds and fore limbs with election of hair, tremors, shivering of
the whole body and respiratory distress, severe convulsions, and respiratory
difficulty prior to death in group-4. The Biochemical parameters showed significant
(P<0.05) increase in the activities of the liver enzymes concomitant with the increase
of the commercial hair dye dosage in the two batches, and decrease in the total
plasma protein levels, albumin, and cholesterol with the increase of commercial hair
dye dosage in the two batches. Hematological parameters showed a significant (p
value <0.05) decrease in complete blood count (associated with significant decreases
in neutrophils and significant increases of lymphocytes) concomitant with the
increasing of commercial hair dye.
1.3.Uses of PPD:-
Paraphenylenediamine is a chemical used mainly in photographic developing
solutions, hair dyes, photocopying and printing ink, black rubber, grease, temporary
tattoos and dark colored cosmetics, hair dye it is used for dying hair and it become
widely mixed with Henna to intensify the black color produced by Henna and also
to reduce the time required for the process.
A substituted form of PPD sold under the name CD-4 is also used as a developing
agent in the C-41 color photographic film development process, reacting with the
silver grains in the film and creating the colored dyes that form the image.
PPD is also used as a Henna surrogate for temporary tattoos. Its usage can lead to
severe contact dermatitis.
PPD is also used as a histological stain for lipids such as myelin. PPD is used by
Lichenologists in the PD test to aid identification of Lichens.(29)
1.4. Acute toxicity:-
The major problem of PPD toxicity results from ingestion of the pure stuff (99%)
for suicidal, homicidal or by mistake. However, there were some cases reported as a
result of topical application during hair dying using pure stuff or while practicing
Henna.
1.5. Treatment of oral toxicity:-
Patients should be monitored for respiratory distress and endotracheal intubation has
to be performed early if there is stridor due to laryngeal edema, or surgical airway
has to be created to prevent hypoxia. There is no specific antidote for PPD, and the
treatment is mainly supportive, it depends on the clinical scenario.
Antihistamines and steroids are commonly used in the management of airway edema
because of the possibility of a hypersensitivity reaction to PPD but there is no
evidence to support this mode of treatment.(30)Alkaline diuresis using isotonic saline,
sodium bicarbonate, and osmotic diuretics are used in the management of
myoglobinuria(31) and trials of PPD removal was attempted using hemoperfusion and
hemodialysis, which had variable results(32). The PPD is not dialyzable(33) , and
dialysis in only supportive therapy.
The most consistent predictor of mortality is amount of hair dye ingested,
hyperkalemia, hypocalcemia and hyperphosphatemia and mortality rates vary
between 0.03% and 60%.(34) Information on the systemic effects and outcome of hair
dye poisoning in pregnant mothers and children are limited.(35)
Awareness about this condition, early diagnosis of rhabdomyolysis and ARF with
timely institution of appropriate supportive management will have a better outcome
in hair dye poisoning. The time of development of renal failure following PPD
intoxication is unpredictable, and hence all patients should be monitored in hospital
for development of renal complications.
Despite the high frequency of cases and mortality, no antidote is available for this
poisoning. (36)
Also hair dye effects on respiratory system result in laryngeal odema, which can be
manage with tracheotomy for saving a life.(37)
1.6. Investigation of PPD:-
MALDI-MS/MS. Matrix-Assisted –Laser-Desorption/Ionization. Tandem mass
spectrometry. Is a method to measure the quantity of PPD concentration in urine
sample, the technique allows the possibility to measure small molecules in large
sample set in a fast, sensitive, precise and accurate manner and to study the
correlation of severity of clinical symptoms and treatment process with urine
concentrations.
This technique provides the possibility to identify the metabolites, mono acetylated
paraphenylene diamine (MAPPD) and diacetylated paraphenylene diamine
(DAPPD) in urine, also the using of HPLC- UV.
There is a new developed wet chemistry assay will allow a simple, sensitive and cost
– efficient test for a fast differential diagnosis for PPD intoxication in hospitals using
urine. (38)
1.7. Safety:-
The aquatic LD50 of PPD is 0.028 mg/L.(39) The U.S. Environmental Protection
Agency reported that in rats and mice chronically exposed to PPD in their diet, it
simply depressed body weights, and no other clinical signs of toxicity were observed
in several studies.(40), the Pub Med search engine found "at least one well-designed
study with detailed exposure assessment" that observed associations between
personal hair dye use and non-Hodgkin's lymphoma, multiple myeloma, acute
leukemia, and bladder cancer,(41) but those associations were not consistently
observed across studies. A formal meta-analysis was not possible due to the
heterogeneity of the exposure assessment across the studies.
1.8. Mechanism of action:-
Paraphenylenediamine is a reducing agent and is thought to be oxidized in vivo to
quinine diamine. It has sharp penetrating odour and produces violent local irritation
of the mucous membranes and skins of sensitive individuals. Quinine diamine is
suggested to be responsible for the sensitization property of PPD. Rhabdomylysis is
considered to be the principle mechanism underlying PPD systemic toxicity. It is in
particular responsible for the renal failure observed in many cases.
1.9. Patch test:-
In 2005–06, it was the tenth-most-prevalent allergen in patch tests (5.0%) (42)
The Centre of Disease Control (CDC) lists PPD as being a contact allergen.
Exposure routes are through inhalation, skin absorption, ingestion, and skin and/or
eye contact; symptoms of exposure include throat irritation (pharynx and larynx),
bronchial asthma, and sensitization dermatitis.(43)(44) Sensitization is a lifelong issue,
which may lead to active sensitization to products including, but not limited to black
clothing, various inks, hair dye, dyed fur, dyed leather, and certain photographic
products. It was voted Allergen of the Year in 2006 by the American Contact
Dermatitis Society. Poisoning by PPD is rare in western countries (45).
1.10. Objective:-
This study was done to assess the hair dye toxicity by using hair dye among
experimental animals (albino rats) in order to investigate the biochemical and
hematological abnormalities, kidney and liver disorder, and their dysfunctions.
2. Materials & Methodology:-
Materials and experimental design:-
2.1. Methodology:-
Two ml of blood samples were collected after slaughtering of each rat after 24 hrs
of last dose in ethylenediamine tetra acetic acid (EDTA) container for
haematological tests and other 2 ml of blood samples were collected in heparinized
containers for biochemical tests. serum was separated by centrifugation at 3000 rpm
for 5 min.
Total proteins, albumin, cholesterol, and the enzyme activities of GOT, GPT, were
measured (liver function test) with specrtophotometer by using commercial kits. and
also urea, creatinine were measured for renal function test.
Determination of hemoglobin concentration (Hb), packed cell volume (PCV), red
blood cells (RBCs) count, mean corpuscular volume (MCV), mean corpuscular
hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH),
were analyzed by a semi-automated hematological analyzer (Sysmex Corporation;
Mundelein, Illinois, Sysmex America, Inc.).
The Animals:-
Forty days-old Albino rats weighted 0.25 kg were obtained from the Faculty of
veterinary medicine, Sudan University of science and technology, and reared in pens
within the premises of the university.
The Experimental design:-
The rats were divided at random into four groups, each of 10 rats. group 1 were the
control and fed normally.
The Materials:-
Solid hair dye was collected from market and beauty homes local markets.
The solid hair dye was grinded into a powder and dissolved in distilled water and
given to rats as follows:-
Orally administered with 10mg/kg body weight of the commercial hair dye (group
2), orally administered with 20mg/kg body weight of the commercial hair dye (group
3), orally administered with 30mg/kg body weight of the commercial hair dye (group
4) for three days.
2.1.1. Clinical chemistry:-
Serum samples were analyzed for the activities of aspartate aminotransferase (AST),
alanine aminotransferase (ALT), and for the concentration of total protein, albumin,
cholesterol, urea and creatinine. 2.1.2. Methods used for determination of
serum constituents:-
Blood samples were collected after slaughtering each rat for biochemical
examination into dry clean and heparinized containers for biochemical tests and
serum was separated by centrifugation at 3000 rpm for 5 minutes.
2.1.3. Liver function tests:-
2.1.3.1. Glutamic oxaloacetic transaminase (GOT/AST):-
Serum glutamic oxaloacetic transaminase activity was measured by
aspectrophotometric method using commercial kit (linear chemicals, Barcelona,
Spain).
Test principle:-
The glutamic oxaloacetic transaminase also called aspartate aminotransferase (AST)
catalyzes the transfer of an amino group from aspartic acid to the alpha ketoglutrate
(α-kg) in a reversible reaction. The end products formed in the reaction are
oxaloacetate and glutamate. The transaminase activity is proportional to the amount
of oxaloacetic acid formed(after adding R1) and measured by a reaction with 2,4
Dinitro phenyl hydrazine (DNPH(R2)) in alkaline solution(R3).
l-aspartate + α-ketoglutrate (AST) ⇆ L-glutamate + oxaloacetate.
Procedure:-
0.5 ml of R1 was mixed with 100 µl of samples in test tubes and incubated for 60
mins at room temperature, Then 0.5 ml of R2 was added and mixure incubated for
20 mins at room temperature, 5ml of R3 was added and solution was incubated for
15 mins at rpm temperature.
The absorbance of sample was read at 505 nm in the spectrophotometer against water
blank and the concentration of samples was calculated from the standard curve.
Serum GOT activity was measured in International unit IU, IU = WU × .0482.
WU (World Unit) of GOT is defined as the amount of enzyme that will form 4.32
× 104 µ mol of glutamate per minute at 25ºc.
2.1.3.2. Glutamic Pyruvic Transaminase (GPT/ALT):-
Serum glutamic pyruvic transaminase activity was measured by
aspectrophotometric method using commercial kit (linear chemicals, Barcelona,
Spain).
Test principle:-
The glutamic pyruvic transaminase also called alanine aminotransferase (ALT)
catalyzes the transfer of an amino group from alanine to the alpha ketoglutrate (α-
kg) in a reversible reaction. The end products formed in the reaction are glutamate
and pyruvate. The transaminase activity is proportional to the amount of pyruvic
acid formed (after adding R1) and measured by a reaction with 2,4 Dinitro phenyl
hydrazine (DNPH(R2) ) in alkaline solution(R3).
l-alanine + α-ketoglutrate (ALT) ⇆ L-glutamate + pyruvate.
Procedure:-
0.5 ml of R1 was mixed with 100 µl of samples in test tubes and incubated for 30
mins at room temperature, then 0.5 ml of R2 was added and mixure incubated for 20
mins at room temperature, 5ml of R3 was added and solution was incubated for 15
mins at room temperature.
The absorbance of sample was read at 505 nm in the spectrophotometer against water
blank and the concentration of samples was calculated from the standard curve.
Serum GPT activity was measured in International unit IU, IU = WU × .0482. WU
(World Unit) of GPT is defined as the amount of enzyme that will form 4.32 × 104
µ mol of pyruvate per minute at 25ºc.
2.1.3.3. Total protein:-
Serum total protein concentration was determined by aspectrophotometric
method using commercial kit (linear chemicals, Barcelona, Spain).
Test principle:-
The method is based on the biuret reaction (R1) in which achelate is formed
between Cu+2 ion and the peptide bonds of the protein in an alkaline solution to
form a violet colored complex. The intensity of color produced is proportional to
the concentration of protein in the sample.
Procedure:-
1 ml of R1 was mixed with 20 µl of sample with use of standard (7g/dl) and
incubated for 10 mins at room temperature.
The absorbance (A) of samples and standards was read at 540 nm in the
spectrophotometer and serum total protein concentration (C) was calculated as
follows:
C (g/dl) = A sample
A standard × 7
2.1.3.4. Albumin:-
Serum albumin concentration was determined by aspectrophotometric method using
commercial kit (linear chemicals, Barcelona, Spain).
Test principle:-
The method is based on the specific binding to the indicator, 3, 5, 5, 5,
tetrabromoceresol, R1 (BromoCeresol Green (BCG)), an ionic dye, and the protein
at acid pH 4.2 with the formation of coloured complex.The intensity of colour
produced is proportional to the concentration of albumin in the sample.
Procedure:-
2 ml of R1 was mixed with 10 µl of sample with use withS standard (5g/dl) and
incubated for 10 mins at room temperature.
The absorbance (A) of samples was read at 630 nm in the spectrophotometer and
serum albumin concentration (C) was calculated as follows:
C (g/dl) = A sample
A standard × 5
2.1.3.5 Cholesterol:-
Serum cholesterol concentration was measured by an enzymatic
spectrophotometric method using commercial kit (linear chemicals, Barcelona,
Spain).
Test principle:-
Cholesterol ester is hydrolyzed enzymatically to free cholesterol and fatty
acid,catalyzed by cholesterol esterase (C.E). In the presence of O2 , the free
cholesterol produced and unstatified cholesterol in the sample is oxidized to
cholestenon and H2O2, catalyzed by cholesterol oxidase (C.O). in the presence of
former the mixture ADPS (N-ethyl-N-propyl-m-anisidine) and 4-aminoantipyrine
(4-AA) are condensed by H2O2,catalyzed by peroxidase to form Quinoneimine
dye. The intensity of colour produced is proportional to the amount of cholesterol
present in serum sample.
Cholesterol ester C.E cholesterol + fatty acids
Cholesterol +O2 C.O cholestenone + H2O2
H2O2 + 4-AA +ADPS peroxidase POD Quinoneimine + 4 H2O
Procedure:-
1 ml of R1 was mixed with 10 µl of sample with use with standard (200mg/dl),
and mixure was incubated for 10 mins at room temperature.
The absorbance (A) of samples and standards was read at 550 nm in the
spectrophotometer and serum cholsterol concentration (C) was calculated as
follows:
C (g/dl) = A sample
A standard × 200
2.1.4 Renal function tests:-
2.1.4.1. Urea Estimation:-
Test principle:-
Urea in the sample originates, of the coupled reactions, a colored complex that can
be measured spectrophotometrically.
Urea + H2O ــــــــurease2 ــــــNH4 + CO2.
NH4 + Salicylate + NaClO ــــــnitroprussideـــــ Indophenol.
Procedure:-
1 ml of R1 was mixed with 10 µl of serum sample with use of standard, mix well
and incubated for 10 mins at room temperature.1 ml of R2 was mixed and
incubated for 10 mins at room temperature.
The absorbance (C) of samples was read at 600 nm aginst the blank in the
spectrophotometer and serum urea concentration (C) was calculated as follows:-
C(mg/dl) = A sample
A standard × Standard × Sample dilution factor.
2.1.4.2. Creatinine Estimation :-
Test principle:-
Creatinine in the sample react with picrate in alkaline medium forming a coloured
complex (Jaffe`s method). The complex formation rate is measured in a short
period to avoid interferences.
Serum samples contain proteins that react in a non specific way,nevertheless, then
result can be corrected subtracting a fixed value. The use of this correction is
known as the Jaff `s method.
Procedure:-
1 ml of R1 was mixed with 0.1 µl of sample with use of standard, mix well and
insert the mixture into the photometer, start stop watch, record the absorbance at
500 nm after 30 seconds (A1) and after 90 seconds (A2) and serum creatinine
concentration in the samples (C) was calculated as follows:-
C(mg/dl) = (A2 –A1) sample
(A2−A1) standard × C standard × Sample dilution factor – correction
factor.
2.2. Haematology:-
Blood samples were examined for haemoglobin concentration (HB), red blood cell
(RBC) count, total white blood cell, packed cell volume (PCV), mean corpuscular
volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular
haemoglobin concentration (MCHC).
2.2.1. Haematological methods:-
Blood samples were collected after slaughtering of each rat after 24 hrs of last dose
into dry clean ethylenediamine tetra acetic acid EDTA containers for
haematological tests.
2.2.2. Haemoglobin concenteration (HB):-
Haemoglobin concentration was determined by a chemical method, in which
Drabkin`s solution was used (0.2g Potassium cyanide, 0.2g potassium
ferriccyanide, and 1g sodium bicarbonate per 1 litter of distilled water).with
spectrophotometer.
Test principle:-
Drabkin`s solution reacts with haemoglobin in the sample to form
cyanomethaemoglobin (colored). The intensity of color produced is proportional to
the amount of haemoglobin concentration in the sample.
Procedure:-
4 ml of Drabkin`s solution were mixed with 20 µl of sample and with use
standard (13.3g/dl), and incubated for 10 mins at room temperature.
The absorbance (A) of samples and standard was read at 520 nm in the
spectrophotometer and haemoglobin concentration was calculated as follows:-
C (g/dl) = A sample
A standard × 13.3
2.2.3. Packed cell volume (PCV):-
Fresh blood samples we
re centrifuged in a microhaematocrit centrifuge (Hawksley and Sons Ltd, England)
for 5 mins. The PCV percentage was read off on the scaling instrument provided
with the centrifuge.
2.2.4. Red blood cell (RBC) count:-
Red blood cells were counted with an improved Neubauer haemocytometer
(Hawksley and Sons Ltd, England). Formal citrate was used as a dilluent.
2.2.5. Mean corpuscular volume (MCV):-
The MCV, in cubic microns, was calculated from RBC and PCV values as follows:-
MCV (m3) = PCV(%) × 10 RBC count in million/m3
2.2.6. Mean corpuscular haemoglobin concentration (MCHC):-
The MCHC was calculated from HB and PCV values as follows:-
MCHC (%) = HB in g/dl × 100 PCV (%)
2.2.7. Mean corpuscular haemoglobin (MCH):-
The MCH was calculated from HB and RBC values as follows:-
MCH (pg) = HB in g/dl × 10 RBC count in million/m3
2.3. Statistical method:-
The significance of differences between means was compared at each time point
using statistical package for social sciences (SPSS) version 11.5 and excel 2007
statistical program. Continuous and categorical variables were analyzed using
student’s test and Chi-square test respectively. P value was considered significant
if it was less than 0.05.
3. Results:-
3.1. Results:-
Groups /
Parameters
Control
Group 1
Group 2(10
mg/kg)
Group 3(20
mg/kg)
Group 4 (30
mg/kg)
GOT (u/l) 38.3 ±2.1 985.5 ±10.3*** 1250.3 ±20.1*** 1400.0 ±18.5***
GPT (u/l) 40.5 ±2.1 130.2 ±1.8*** 252.6 ±7.7*** 262.0 ±15.5***
T. proteins
(g/dl) 7.3 ±0.5 6.8 ±0.6 6.4 ±0.3 6.0 ±0.4*
Cholesterol
(mg/dl) 90.5 ±14.4 70.9 ±3.1*** 68.3 ±3.4** 69.1 ±4.1*
Albumin
(g/dl) 4.4 ±0.6 4.26 ±0.4 3.55 ±0.3* 3.19 ±0.4*
* = P<0.05; ** = P<0.01; *** = P<0.001
Table (3.1):The mean differences of Biochemical parameters of serum samples
collected from rats that received three oral doses (10-20-30 mg/kg b.w.), of
PPD for 3 day.
Figure (3.1) showing (GOT concentration) differences of PPD toxicity.
Figure (3.2) showing (GPT concentration) differences of PPD toxicity .
0
200
400
600
800
1000
1200
1400
1600
Control Group 1 Group 2(10 mg/kg) Group 3(20 mg/kg) Group 4 (30 mg/kg)
GOT (u/l)
0
50
100
150
200
250
300
Control Group 1 Group 2(10 mg/kg) Group 3(20 mg/kg) Group 4 (30 mg/kg)
GPT (u/l)
Figure (3.3) showing (T.protein concentration) differences of PPD toxicity.
Figure (3.4) showing (Cholesterol concentration) differences of PPD toxicity.
0
1
2
3
4
5
6
7
8
Control Group 1 Group 2(10 mg/kg) Group 3(20 mg/kg) Group 4 (30 mg/kg)
T. proteins (g/dl)
0
10
20
30
40
50
60
70
80
90
100
Control Group 1 Group 2(10mg/kg)
Group 3(20mg/kg)
Group 4 (30mg/kg)
Cholesterol (mg/dl)
Figure (3.5) showing (Albumin concentration) differences of PPD toxicity.
0
1
2
3
4
5
Control Group 1 Group 2(10 mg/kg) Group 3(20 mg/kg) Group 4 (30 mg/kg)
Albumin (g/dl)
Groups/
Parameter
Control
Group 1
Group 2(10
mg/kg)
Group 3 (20
mg/kg)
Group 4 (30
mg/kg)
Hb (g/dl) 12.5±0.55 10.24±0.63* 9.34±0.56* 8.0±0.52**
PCV (%) 40.4±0.34 30.6±0.64* 25.00±1.64*** 23.65±1.57***
RBCs˟663/CM
M
5230.00±550.0
0
4950.00±115.0
0*
3955.00±110.80
**
3445.00±123.50*
**
MCH (pg) 27.85±2.1 19.95±2.30* 17.50±1.68** 15.75±2.10***
MCV (fl) 85.73±4.26 66.00±2.90*** 58.83±5.10*** 51.50±3.60***
MCHC (g/dl) 32.00±1.53 38.10±2.30 43.18±2.70* 46.70±2.60**
*=P<0.05; **=P<0.01; ***=P<0.001
Table ( 3.2):The mean differences of Haematological parameters of blood
samples collected from rats that received three oral doses (10-20-30 mg/kg b.w.),
of PPD for 3 day.
Figure (3.6) showing (PCV concentration) differences of PPD toxicity.
Figure (3.7) showing (HB concentration) differences of PPD toxicity.
0
5
10
15
20
25
30
35
40
45
Control Group 1 Group 2(10 mg/kg) Group 3 (20 mg/kg) Group 4 (30 mg/kg)
PCV (%)
0
2
4
6
8
10
12
14
Control Group 1 Group 2(10 mg/kg) Group 3 (20 mg/kg) Group 4 (30 mg/kg)
Hb (g/dl)
Figure (3.8) showing (RBCs concentration) differences of PPD toxicity .
Figure (3.9) showing (MCH concentration) differences of PPD toxicity.
0
1000
2000
3000
4000
5000
6000
Control Group 1 Group 2(10mg/kg)
Group 3 (20mg/kg)
Group 4 (30mg/kg)
RBCs˟103/CMM
0
5
10
15
20
25
30
Control Group 1 Group 2(10 mg/kg) Group 3 (20 mg/kg) Group 4 (30 mg/kg)
MCH (pg)
Figure (3.10) showing (MCV concentration) differences of PPD toxicity.
Figure (3.11) showing (MCHC concentration) differences of PPD toxicity.
0
10
20
30
40
50
60
70
80
90
100
Control Group 1 Group 2(10 mg/kg) Group 3 (20 mg/kg) Group 4 (30 mg/kg)
MCV (fl)
0
5
10
15
20
25
30
35
40
45
50
Control Group 1 Group 2(10 mg/kg) Group 3 (20 mg/kg)Group 4 (30 mg/kg)
MCHC (g/dl)
Groups/
Parameter
Control
Group 1
Group 2(10
mg/kg)
Group 3 (20
mg/kg)
Group 4 (30
mg/kg)
Urea
(mg/dl) 26.50±3.23 38.4±1.5* 40.53±2.15* 42.61±2.35**
Creatinine
(mg/dl) 0.77±0.07 0.96±0.066* 0.98±0.051*** 1.15±0.052***
*=P<0.05; **=P<0.01; ***=P<0.001
Table (3.3): The mean differences of Biochemical parameters of serum
samples collected from rats that received three oral doses (10-20-30
mg/kg b.w.), of PPD for 3 day.
Figure (3.12) showing (Urea concentration) differences of PPD toxicity.
0
5
10
15
20
25
30
35
40
45
Control Group 1 Group 2(10 mg/kg) Group 3 (20 mg/kg) Group 4 (30 mg/kg)
Urea (mg/dl)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control Group 1 Group 2(10 mg/kg) Group 3 (20 mg/kg) Group 4 (30 mg/kg)
Creatinine (mg/dl)
Figure (3.13) showing (Creatinine concentration) differences of PPD toxicity.
4. Discussion & Recommendations:-
4.1. Discussion:-
This study was done to assess the hair dye toxicity by using commercial hair dye in
a way to estimate the hazards of this dye on rats , since it is known that toxic
effects in humans are usually in the same range as those of experimental animals .
PPD is the main constituent in hair dye and is an organic derivative of
paranitroaniline , when ingested in a dose-dependent manner, results in severe
hypersensitivity (itching, angioedema, asphyxia) and rhabdomyolysis (paresis of
extremities, cola-colored urine , oliguria ,markedly elevated creatinine, urea, AST
and ALT). Other features such as anemia, leukocytosis, hemoglobinemia,
haemoglobinurea, and liver and kidney dysfunction have been reported . In animal
model, PPD induces rhabdomyolysis, followed by continuous contraction and
irreversible structural changes in the muscles , in this study.
The commercial hair dye was introduced in this study through oral routes. As
shown in the results ,oral ingestion results in a direct effect and toxicity of
commercial hair dye. At higher doses of the commercial hair dye, there was broad
deviation from the normal values in biochemical (liver function tests & renal
function tests) and haematological parameters compared to lower doses of hair dye
in all groups, because the concentration of a toxic agent is increased.
Our results showed significant increase in liver enzymes (GOT, GPT) activities in
a different doses, also significant increase in renal parameters (Urea , Creatinine)
activities in adifferendoses and there is a decrease in the total protein levels,
albumin, and cholesterol associated with the increase of the commercial hair dye.
Our finding is in agreement with other studies , when their administration of PPD
to rats revealed a significant increase in GOT, GPT, urea , creatinine and a
significant decrease in total proteins and albumin and showed significant increase
among different doses of oral ingestion administration of commercial hair dye
compared with the control groups. Our result showed consistency with other
studies that find toxic effect to the liver and kidney . pointed out by the substantial
leakage of enzymes
contained in the cells of hepatic tissues to the blood. It has been reported that low
cholesterol level is usually associated with hepatocellular damage. The decrease in
the level of cholesterol in our study may be associated with hepatic lipidosis and
obstructive liver diseases.
The haematological investigations showed significant decrease in Hb and PVC
values which may be attributed to the escape of plasma from circulation to the
surrounding tissues, in addition to significant decreases in RBCs, MCH and MCV
values. These haematological changes indicate that, anemia may occur as a result
of exposure to commercial hair dye orally. The possible cause for anemia is the
hemolytic effect of PPD on RBCs , anemia was noticed in rats that received high
doses of the commercial hair dye.
Clinical features were shown in all rats administered orally with the commercial
hair dye, however, the clinical features rate from slight weakness in group 2 which
expose to 10 mg/dl to neck, and pharyngeal oedema in group-3 which expose to 20
mg/dl up to severe weakness in hinds and limbs with falling of hair, tremors, and
respiratory distress, and there were severe convulsions and respiratory difficulties
before death which occurred at about three to four hours after oral ingestion of the
commercial hair dye in group-4 which expose to 30 mg/dl.
As seen in Table 1 and Table 3, the biochemical parameters showed significant
(P<0.05) increase in activities of the liver enzymes glutamate oxalotransferase
(GOT), glutamate pyruvate transferase (GPT), urea , creatinine and there is a
decrease in the total plasma protein levels , albumin , and cholesterol when
compared with the control group.
The haematological parameters showed significant (P<0.05) decrease in the level
of HB, RBCs count, PCV, MCV, MCH and increase at the level of MCHC when
compared with the control group as seen in Table 2.
4.2. Recommendations:-
After discussion of all these results and comparison with the normal results that
taken from control group, these results and its comparisons give us these
recommendations:-
- Paraphenylenediamine (PPD) toxicity is a major health problem, in Sudan ,
Africa , and Middle East with negative impacts on the limited health care
resources.
- The official authorities in Sudan should restrict the use of trade of PPD in a
beauty local markets, because of its highly hazardous .
- A PPD formulation with concentrations less than 6% (the allowable concentration
according to the European standard) can replaced by available commercial PPD to
be mixed with henna for cosmetic purposes .
- Extensive information, public programmes and the collaboration of different
health authorities with the communities to rise the public awareness of PPD
related hazards .
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