Foods are essential to remain healthy. Very often our food is contaminated, unhealthy and adulterated in various ways. Adulteration in food occurs when substances are either added or substituted. Adulteration of foods can either be deliberate, accidental or innate. However, it has been found that unscrupulous traders add adulterants to food items for their own benefits. Hundreds of years ago, the Greek utilization of adulterants for profitable purposes in certain items has been reported. In fact the shift from agricultural to industrial society has led to adulteration of food products to grow significantly.  In the past few years, more and more cases of food adulteration have been reported and it represents a serious threat to the human health. 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]. 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.
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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. Furthermore, food industry endeavours to offer visually attractive foods that have excellent taste and meet up the consumer's requirements on quality and cost [Mukul, 2007]. 
Adulteration has continued in recent times, with several remarkable instances involving the spice industry. In the past decade, adulteration of food such as spices and chilli products with industrial dyes such as Sudan red and other unapproved dyes has become one of the serious problems in many countries. These illegal dyes are being used by certain manufacturers 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. The European Union and the United States has banned the utilization of these colours 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 colour of chilli powders and bell pepper in various countries. 
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) [EFSA, 2005]. 
In October 2004, the governmental chemical institute in Wuppertal, Germany reported the identification of unapproved colour such as Para Red in curry and bell pepper powder. A series of notifications regarding the presence of Para Red in chilli powder, paprika powder and many other spices as well as rhodamine B were detected in chilli 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].  A survey carried out in Lanzhou City of China for the determination of Sudan dyes on 107 chilli products in beef noodles samples found that 15 samples were detected to be adulterated with Sudan I or IV with levels ranging from 2.18 to 110 mg/kg [Wang et al., 2010]. 1http://en.cnki.com.cn/Article_en/CJFDTOTAL-ZWJZ201002069.htm journal 11.12.13 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.
1.3 Food Adulteration legislation
Always on Time
Marked to Standard
"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: Unauthorized 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 colouring 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 colouring substances in foodstuff in countries like Mainland China, European Union, Australia and Canada. 
In South Africa, the colourant is forbidden for utilization in foodstuffs by the set of laws "Relating to Food Colourants (R.1008) of the Foodstuffs, Cosmetics and Disinfectant Act 54 of 1972" [health24, ca.2007]. 
1.4 Government action towards food adulteration
Prevention of Food Adulteration Act, 1955 of India state that this harmful threat of food adulteration is required to be argued against by making the lawful provisions stricter and deterrent even leading to life imprisonment for adulterations which causes severe hurt and menace to human being. This negligence is furthermore being dealt with through efficient health education method. 
A campaign was hold from July to August 2011 in Bangladesh against the adulteration of food products after considering the threat, to ascertain rights of consumers, to put in force court activity and exemplary penalty to deceitful traders. 
Furthermore there are rules and regulations on adulteration of foodstuffs which legalise. Some extremely severe measures including capital punishment are in position; however these might not fright the adulterators ahead of their deceitful practices. Nevertheless, on top of raising mass consciousness through media campaigns about the risks of consuming unsafe foods, public would have to be encouraged to search for recourse to seek redress for their injustice [The Daily Star, 2012]. 
1.5 Adulterants present in spices and chilli products
Food items may be adulterated in various ways to increase the appearance of the products by adding inferior substances which in turn enhance profit margin. There different types of food adulteration; for instance bakers from time to time added alum and chalk to the flour leading to whitening of bread.
A brief review of some common illegal dyes used as adulterants added in spices and chilli products 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 [Gresshma & Reject, 2012].  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 [Gresshma & Reject, 2012].  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 [EFSA, 2005].  According to OSHA the intravenous LD50 in rats is 89.5 mg/kg. 
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An article published by Kochi (2007) in THE FINANCIAL EXPRESS, these cancer-causing Rhodamine B dye is largely used as marker and as pesticide sprayer in paddy fields and chilli farms.  Rhodamine was also detected in pastes and curry powder. Futhermore, the toxic dyes were also found in dried chillis in Hunan, China in year 2012 with the intention of improving its appearance which was in turn confiscated. According to the Consumers Association of Penang (CAP), Food manufacturers are still using rhodamine dye in the preparation of belacan (shrimp paste) and several popular Chinese buns and kuih [thestar online, 2007]. 
126.96.36.199 Analysis of Rhodamine Dyes
Shanshan et al., (2010) developed immunoassay and a sol-gel-based immunoaffinity chromatography (IAC) purification method for the determination of Rhodamine B in chilli powder such that the detection limit of the ELISA method was 1ng/g. The recovery for the IAC method was 68.1-86.2% at 1 ng/g and 72.6-89.3% at 5 ng/g in a spiked chilli powder. The Fortified samples were examined by HPLC-MS after IAC purification, and the results demonstrated a good conformity between the two techniques. The ELISA could be a suitable tool for screening Rhodamine B residue in foodstuffs, and the IAC cleanup procedure coupled with HPLC-MS could be an efficient alternative technique for the determination of Rhodamine B in a variety of substances.
Rhodamine can be detected using Visible Spectrophotometry (UV). Gresshma & Reject (2012) analysed sweets and confectionery collected from street foods in India. 60 samples out of 75 samples analysed were found to contain Rhodamine. The concentration of the extracted Rhodamine B from the samples varied between 0.071 to 1.09Î¼g/ml. 
In another study carried out in India; most precisely in Hyderabad determine the type of colour added in 545 ready-to-eat foods was carried out. Permitted colours were detected in 90 percent while unacceptable colours were detected in 8 percent of the samples. Amongst the unacceptable colours detected in the samples, rhodamine was most frequently utilized [Jonnalagadda et al., 2004]. 
YIN et al., (2012) used Solid Phase Extraction-High Performance Liquid Chromatography-coupled with Mass Spectrometry (SPE-HPLC-MS/MS) technique for determining the presence of Rhodamine dye in spices. Samples were extracted using acetonitrile, centrifuged, purified and passed through an ion exchange SPE column after the addition of 10 Ml 1% trichloroacetic acid. The resulting analyte was analysed using HPLC equipped with a C18 column of length 100 mm and internal diameter of 2.0 mm using methanol and 0.1% formic acid solution as mobile phase. Electrospray ionization in positive ion mode-MS/MS in multiple reactions monitoring (MRM) mode was used as detector. The LOQ obtain in the spice sample was 1.2 Î¼g/kg. 
1.5.2 Sudan dyes
Sudan dye is another class of 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 added in cosmetics and medicines applied superficially while Sudan IV can be used in veterinary and human remedy as an cream or dressings to stimulate wound healing. 
Table 1: Sudan dyes
IUPAC Name/ Formula
Reddish brown crystals
1-(2-methyl-4-(2-methylphenyldiazenyl) phenyl) azonapthalen-2-ol
Sudan Red B
Sudan Red 7B
Sudan Red G
This is the chemical structure of para red.
Sudan Orange G
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 [health24, ca.2007].  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)
According to a report published by EFSA's Scientific Panel, it has been found that the Sudan dyes (Sudan I, II, III, IV, Para Red) except for Orange II may be considered to be both carcinogenic and genotoxic. While Orange II is perhaps genotoxic, information is missing to find out whether or not this particular dye is a potential carcinogen. The Opinion provides a comprehensive toxicological analysis for every dye, based on the carcinogenicity, genotoxicity and chemical similarities. Furthermore, the EFSA's Deputy Executive Director and Director of Science give explanation that a full risk assessment of these particular illegal dyes due to lack of data. Nevertheless, the evaluation performed by the Panel of the inadequate toxicological information proves that the suspected carcinogenic and or genotoxic possibility of those dyes which Member States and the Commission had previously considered unauthorisable to be used in foodstuffs [EFSA, 2005]. 
Para Red is structurally similar to other dyes such as Sudan I, however information are very little. As these dyes show their genotoxic and carcinogenic effects merely after metabolic activation it is probable that alterations of the structure may change those potentials however in the absence of information it would be practical to presume that Para Red is potentially genotoxic and probably carcinogenic
The acute toxicity of Orange II dye is characterized by the induction of methaemoglobinaemia as well as an increased red blood cell turnover. Genotoxicity in bacterial investigations has not been confirmed; however these test systems are incomplete in an appropriate azo-reduction step. Positive effects have been observed in one vivo study and in one vitro mammalian cell analysis, even though at extremely high dosage, suggest that activation to a genotoxic metabolite could arise in mammalian systems. 
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 chilli 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. 
In a study, [Shaofeng et al., 2007] developed a new method for analysis of four Sudan dyes in hot chilli by pressurized CEC with amperometric detection using silica packed columns, monitored using carbon electrode. The limit of detection for the four Sudan dyes ranged from 8.0 Ã- 10-7 to 1.2 Ã- 10-6mol/L. To evaluate the feasibility and reliability of this technique, the proposed pCEC-AD method was further demonstrated with hot chilli samples spiked with Sudan dyes.
A report published by CAMAG (2009) which uses reversed phase High Performance Thin Layer Chromatography for the determination of illegal dyes in chilli, curry and paprika. Samples were extracted using acetonitrile, purified using SPE cartridge, evaporated to dryness, applied on HPTLC plates RP18F254s and develop in appropriate tank using acetonitrile: 25% ammonia (19:1) as solvent system. The resulting plate was analysed by using TLC visualiser documentation system. The limit of detection for Sudan I in curry and paprika was 5mg/kg by visual analysis. However, using densitometry the LOD was between 1 to 3mg/kg. 
Krishnamacharyulu & Garimella (2010) published Sudan dyes level detected in 6 six spice products obtained from different countries such that, three samples were found to be contaminated with Sudan I and one sample with Sudan IV. 
Table: 2 Sudan dyes level observed in spice products obtained from different countries
Name of product
Mixed spice powder
1.5, 3, 1063
Macro-fingerprint characteristic of infrared spectroscopy was used to identify and quantify the presence of Sudan dyes in paprika red, such that the limit of determination was nearly one percent [Zhang et al., 2012]. 
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 chilli 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. 
In this present study, Rhodamine B, Sudan I, Sudan II, Sudan III and Sudan IV dyes and any added starch were analysed in nine mixed spices and one chilli sauce originating from Mauritius as well as from other countries by using Thin Layer Chromatography, Ultra Violet Visible Spectroscopy and IR spectroscopy.