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Foods are essential to remain healthy. Moreover, it has been found that, in the past few years, there are more and more cases of food adulteration in almost every part of the world such that, food adulteration has become one of the serious problems. Most of our food is contaminated, unhealthy and adulterated in various ways. Traders no longer consider the harmful effects of their acts to human's health such that they thought about their own benefits. Adulteration of foods can either be deliberate, accidental or innate. Nowadays, it is hard for the consumer to identify food products which are safe due to unreliable advertisements and food adulteration.  (Gupta et al., (2009))
Let us defined the word "adulteration"; adulteration in food is generally present in its most basic form, banned substances that are either added or substituted. The addition of industrial dyes in food products constitutes adulteration of foodstuffs as these substances are not allowed to be added as food colours. The presence of adulterants in food is prohibited by regulation, custom and practice; thus making the food impure by adding inferior or less desirable materials or elements. 
The most common practice is the intentional addition of an adulterant to a food in order to increase the value of the food through deception. Adulteration of food is also performed both to increase profit or due to negligence and lack in proper hygienic condition to process, store up, transport and sell.  Another reason is competition between food companies and industries.
In the past few years, adulteration of food such as spices and chilli products with Sudan red and unapproved dyes has become one of the serious problems in many countries, such that these illegal dyes are being used in minimum amount by certain manufactures to enlarge the profit margin or to compete against other food industries. Research on Cancer showed that the International Agency classified these azo dyes as class three human carcinogens. Due to this information the European Union and the United States do not authorize the utilization of these colors that is Sudan dyes and rhodamine as food-additives. Sudan dyes are not allowed at any level in foods. Nevertheless, these dyes are still being used so as to intensify the color of chili powders and bell pepper in various countries. 
Hundreds of years ago, the adulteration of food products was initially found by the Greek utilization of adulterants for profitable purposes in certain items. The shift from agricultural to industrial society leads to adulteration of food products to grow in significance. 
Adulteration has continued in recent times, with several remarkable instances involving the spice industry. In Hungary, ground paprika lead oxide was added as adulterant in 1994, and in May 2003, ground capsicums in India were found to contain Sudan I at a level of 4000ppm. Ever since, several EU Member States have send notifications through "the Rapid Alert System for Food and Feed (RASFF)" showing the presence of Sudan I, II, III, IV in curry and chilli powder, curry and chilli based processed foodstuffs. In general, the sources of contaminated processed foodstuffs were detected within the European Union, although the prime source is considered to be the utilization of contaminated ingredients from other countries (RASFF, 2005). In June 2003, an evaluation was carried out to control the unauthorized use of Sudan I in powdered chilli and chilli based products (Commission Decision 2003/460/EC). 
In October 2004, the governmental chemical institute in Wuppertal, Germany reported the identification of unapproved color such as Para Red in curry and bell pepper powder. A series of notifications regarding the presence of Para Red in chili powder, paprika powder and many other spices as well as rhodamine B were detected in chili powder in February 2005.Thus, so as to prevent the use of prohibited dyes in food, the European Commission raised the alertness of the food industry at EU level with reverence to their responsibilities under the food.  More than six hundred food products such as pizza, noodle soup, fish sauce and Worchester sauce have been recalled to contain Sudan dyes in United Kingdom.  ( Chailapakul et al.,(no date))
According to a survey carried out in Mauritius in year 2011,roasted peanuts were found to be coloured with the illegal dye rhodamine and in year 2012, certain spices mix of a specific Brand were found to be adulterated with Sudan dyes and research are still being performed to identify the cause of the addition of these illegal dyes.
1.2 Illegal dyes present in spices and chilli products
A brief description of illegal dyes, namely rhodamine and Sudan dyes giving their effects on the human body are given.
Rhodamine often used as a dye and laser gain medium is a family of related chemical compounds, known as fluorone dyes. Rhodamine is used as a tracer dye within water determining the rate, direction of flow and transport. Rhodamine dyes are usually poisonous, and generally soluble in water, ethanol and methanol.  Since rhodamine dyes fluoresce they can be easily detected by using fluorometers as instrument.
Rhodamine dyes are widely used in biotechnology applications like fluorescence microscopy, fluorescence correlation spectroscopy, flow cytometry, and ELISA.
Figure 1: Structure of Rhodamine 
22.214.171.124 Toxicity of Rhodamine Dyes
In general, Rhodamine dyes are considered to be toxic. As per PFA Act, 1954, Rhodamine is a banned dye.  Tests have been carried out on rats and mice by subcutaneous and by oral administration indicating that the commercial dye, rhodamine B was mutagenic in rats after activation in vitro systems, and after injected subcutaneously, producing local sarcomas. However, according to Opinion of the Scientific Panel on Food Additives (2005), it has been found that this effect might possibly be due to unidentified impurities.Positive result was detected in Drosophila melanogaster which regard Rhodamine B to be genotoxic. Thus we can conclude that based on these information, Rhodamine B can be both genotoxic and carcinogenic.  The intravenous LD50 in rats is 89.5 mg/kg. 
126.96.36.199 Analysis of Rhodamine Dyes
A study carried out by Gresshma & Reject (2012) using Visible Spectrophotometry method reported that, sweets and confectionery collected from street foods in India, 60 samples out of 75 samples analysed demonstrated positive outcomes. The concentration of the extracted Rhodamine B from the samples varies between 0.071 to 1.09Î¼g/ml. 
A case study was carried out in India; most precisely in Hyderabad to examine the type of color added in 545 ready-to-eat foods among those are colored synthetic powders and sugar toys. After investigation, permitted colors were detected in 90 percent and unacceptable colors were detected in 8 percent of the samples. Amongst the unacceptable colors detected in the samples, rhodamine was most frequently utilized.  (Jonnalagadda et al., (2004))
Hoenicke (2006) used high performance liquid chromatography coupled with mass spectrometry to determine the level of Sudan I and IV in chili and turmeric in six countries namely India, Italy, Netherlands, United Kingdom and Turkey. Less than 1ppm of Sudan I and IV were detected. Sudan I levels were reported to vary between 10 to
1.2.2 Sudan dyes
Sudan dyes are synthetic chemical azo dyes with general structure R1-N=N-R2. Sudan dyes have relatively high molecular weights and low polarity which make them soluble in oil  (Stuart 2006). However these dyes are "hydrophobic molecules".  A study by American Spice Trade Association (2005) published that these oil-soluble dyes are legally used in the leather and fabric industries. Sudan dyes are readily available and are quite inexpensive. Sudan I, II, III, IV are red dyes used to colour mineral products such as oils, waxes, petrol, hydrocarbon solvents, plastics, shoe and floor polishes.  Sudan II and III can be used in cosmetics and drugs applied externally whereas Sudan IV can be used in veterinary and human medicine as an ointment or dressings for stimulating wound healing. 
Table 1: Properties of Sudan dyes
IUPAC Name/ Formula
Reddish brown crystals
1-(2-methyl-4-(2-methylphenyldiazenyl) phenyl) azonapthalen-2-ol
188.8.131.52 Toxicity of Sudan dyes
Based on toxicological evidence in 1973, "Joint Food and Agriculture Organization /World Health Organisation Expert Committee on Food Additives (JECFA)" considered Sudan I to be unsafe to be use in food. Even though Sudan dyes have been accounted as contact allergens and sensitisers, the utmost concern has been on the possibility to be carcinogens. In 1975, evaluation by the "International Agency for Research on Cancer (IARC)" on Sudan dyes found that following its subcutaneous administration, Sudan I was carcinogenic in mice, giving rise to liver and bladder tumour. Evaluation carried out in 1987 considered that there was insufficient information for cancer causing in humans. According to some research, it has been found that, Sudan I-IV is divided into amines in the body and some of these amines may be classified to be carcinogens.  Futher research has been carried out showing that the azo dyes is converted into a specific form which in turn affect DNA of cells in the body causing harm which can be passed on to the next generation of cells in the affected tissue, eventually leading to cancer.  (BBC news)
184.108.40.206 Analysis of Sudan dyes
Hoenicke (2006) used high performance liquid chromatography coupled with mass spectrometry to determine the level of Sudan I and IV in chili and turmeric in six countries namely India, Italy, Netherlands, United Kingdom and Turkey. Less than 1ppm of Sudan I and IV were detected. Sudan I levels were reported to vary between 10 to 120 Âµg/kg in oleoresins and Sudan IV level in paprika powder was in the range of 10-20 ppb. 
Krishnamacharyulu & Garimella (2010) published Sudan dyes level detected in spice products obtained from different countries. 
Table: Sudan dyes level observed in spice products obtained from different countries
Name of product
Mised spice powder
1.5, 3, 1063
Liu & Gong (2012) used high performance liquid chromatography (HPLC) with on-line photochemical derivatization and fluorescence detection to measure Sudan I, II, III, B in chili oil such that the limits of detection (LODs) range between 0.009 to 0.054 ppm and the limits of quantification (LOQs) range between 0.030 to 0.181 ppm. 
1.4 Food Adulteration legislation
"The Prevention of Food Adulteration Bill was passed by both the house of Parliament and received the assent of the President on 29th September, 1954. It came into force on Ist June, 1955 as THE PREVENTION OF FOOD ADULTERATION ACT, 1954 (37 of 1954)".
Section 23 of the Food Adulteration Act 1954 of the Republic of India reads as follows: Unauthorised addition of colouring matter prohibited :- The addition of a colouring matter to any article of food except as specifically permitted by these rules, is prohibited.
In Hong Kong, Sudan dyes are unacceptable as coloring substances in food under the Colouring Matter in Food rules made under the "Public Health and Municipal Services (Cap.132)".
Sudan dyes are not permitted to be used as coloring substances in foodstuff in countries like Mainland China, European Union, Australia and Canada. 
In South Africa, the colorant is forbidden for utilization in foodstuffs by the set of laws "Relating to Food Colorants (R.1008) of the Foodstuffs, Cosmetics and Disinfectant Act 54 of 1972". 
1.5 Aim and Objectives
The aim of this study is to determine the presence of illegal dyes in chilli products and mixed spices.
The main objectives are:
To determine the presence of illegal dyes such as rhodamine and Sudan dyes
To test for the presence of added starch