CHAPTER 6 DISCUSSION

Transcription

1 CHAPTER 6 DISCUSSION 119

2 1. CHEMICAL AND BIOCHEMICAL ASPECTS OF FOOD COLOURANTS Dietary pattern provides the background information to determine the maximum permitted level (MPL) for the approval for use of a chemical or an additive in food (Miguel, 1998). Food intake studies are carried out for the formulation of nutrition policy and food chemical intake assessment (Gibney, 1999). The intake data forms the base line information for implementing monetary programmes to update the available knowledge on the safety of exposure to potentially harmful substances in foods (Turriny et al, 1991; Gilsenan et al, 2003). Although the additives are required as technological necessity, a constant concern about the potential risk associated with exposure to these chemicals in the diet exists. The safety assessment of approved food additives are ensured by national and international bodies such as FAO/WHO, FDA- USA, JECFA and CAC. In India the use of food additive is regulated and controlled by the Prevention of Food Adulteration (PFA) Act. Colours form a major class of food additives in India. Many location based studies on the pattern of use of food colours have been conducted which show product and specific variation in the use of colours (Padmaja et al, 2004; Pratima et al, 2004; Tripathi et al, 2007). The indiscriminate use of permitted and non permitted colours was shown to induce wide range of reactions in humans and animals. The usage of permitted colours has also evoked concern because they are being used in excess of the statutory limit of 100 ppm. The safety of repeated exposure to a permitted synthetic colourants may also result in severe toxicity as 120

3 they are being used far in excess of the permissible limit of 100 ppm (Pratima and Sudershan, 2008). When a risk assessment is being undertaken, the outcome is the derivation of the Average Daily Intake (ADI). The ADI refers to the amount of food additives that can be taken daily in diet, over a lifetime without any risk of ill and the value serves as an upper limit. The calculation of ADI is followed by the authorization of the use of different foodstuffs and their potential intake. Exposure assessment to food colours in a selected population was attempted by Pratima et al (2004). The calculation of ADI is followed by the use of additives and their potential intake. Many of the developed countries have undertaken national level surveillance to collect information of food additives intake (Fondu, 1992; FSA, 2000; Leclercq et al, 2000; Nzfsa, 2008). However no such attempts have been undertaken in many developing countries including India. In India the use of food additives is on the increase. The trend of consumption of foods coloured with synthetic dyes has been increasing over the years. The global production of food dye is 8000 tons per year and India contributes 2% of world production (Das and Mukherjee, 2004). They are used in a wide variety of foods such as beverages, dairy products, cereals, bakery goods, snack foods and ice - creams. Unfortunately no baseline data is available either on the pattern or on the quantity of these chemicals being consumed by an average Indian. India has no ADI and hence the PFA has recommended the use of colourants to be limited to 100 ppm for the majority of the food items irrespective of the nature of colours or the type of foods in which they are being added. 121

4 Ladu, Jilebi and Halwa are the three common and major sweet items which are being consumed by all people irrespective of age, sex and socio-economic status. These food items are available in different colours and the general public consuming them have no idea about the colourants used and the concentration of the colourants in these food items. There is also a belief that all the colours being used in foodstuffs need not be from the list of permitted colourants. Hence this study was attempted to isolate, characterize and quantify the colourants being used in the above food items. Out of the one thousand samples collected for the study unpermitted colours were not detected in any of the samples. This is contrarary to the earlier reports that more than 20% of the food items contain unpermitted synthetic colourants (Khanna et al, 1987; Pratima et al, 2003; Padmaja et al, 2004). The finding clearly shows that the use of unpermitted colours is not being practiced in this part of India may be because of the high literacy rate and awareness among the public about hazardous effect of non-permitted colours. The quantitative estimation of the colourants using spectrophotometric technique reveals that 75% of the sampled contained synthetic food colourants within the permissible limit of 100 ppm. Remaining 25% samples contained colourants above 100 ppm. Nine percentage of Ladu and 10% of Jilebi samples contained more than 200 ppm (more than double the permissible limit) of the colourants whereas in 14% of the Halwa samples the colourants were present above the level of 200 ppm. All the remaining samples had colour concentration in between 100 and 200 ppm. Earlier studies conducted by Padmaja et al (2004) had shown that more than 73% of all the food samples collected contained colourants above the adulteration limit. Similar findings were also reported by 122

5 Sawaya et al (2007) who studied the consumption pattern of artificially coloured food in Kuwait children. Dixit et al (1995) showed that sunset yellow FCF and tartrazine were most popular among people of Utter Pradesh, India. The intake of these dyes exceeded the ADI for children and adolescents. They also reported that metanil yellow and rhodamine B (two unpermitted colours) were encountered in nearly 10 % of the total 478 samples collected. The present study is not in agreement with the above studies as no unpermitted colours were detected in any of the thousand samples analyzed. Moreover in the present study more than three fourth of the samples contained colourants within the PFA limits, only 25% of them contained colourants above 100 ppm. A very small portion of the samples contained colourants above 200 ppm. In this study out of the eight colours permitted by the PFA Act, only four colours and their combinations were detected in the sweet samples. The synthetic colours isolated were tartrazine (Yellow), sunset yellow (Red), carmoisine (red) and brilliant blue (blue). Orange colour was obtained by mixing tartrazine and sunset yellow and green colour by mixing tartrazine and brilliant blue. Brown colour was obtained either by mixing tartrazine, carmoisine and brilliant blue or by mixing sunset yellow, carmoisine and brilliant blue. The predominant use of tartrazine and sunset yellow in common food items was reported earlier by Pratimna and Sudershan (2008). Our findings are well in agreement with the earlier study as we also observed these two colours in majority of the food items. 123

6 Brown and green colours were the favoured colour of Halwa in Kozhikode, the area from where the samples were collected. The total concentration of the colourants was found to be higher than 200 ppm in 18 % of the present green or brown samples. Similar studies on the concentration of the colourants in colour combinations are scanty and inconclusive from this area. According to Pratima and Sudershan (2008) some foods contained combinations of colours in order to bring desired shade to the food. Brillint blue and carmoisine were detected by them in most of the colour combinations which is in agreement with the present finding. Tripathy et al (2010) also reported similar findings. The intake of colours in the majority of the developed countries is invariably much lower than the ADI. In countries like Brazil, Switzerland, Italy and Finland the use of the permitted dyes varied from 1-26% of the ADI (Pentilla, 1996). In India majority of the studies have revealed the use of non-permitted colours as they were available at cheaper price (Padmaja et al, 2004; Tripathy et al, 2007). Concentration of colourants 20 times above the permissible limit has been reported earlier. In the present study the majority of the food items contained colourants within the permissible limit. Tripathy et al (2010) also reported the use of synthetic colourants above the permissible limit. They have reported that the colour concentration in food materials varied from season to season and in various food items. In this study no attempts were made to compare the colour concentration in different seasons and the study was limited to three sweet items. 124

7 The present result is not in agreement with the earlier findings in two aspects. 1) In the present series unpermitted colourants were not encountered in any of the samples and 2) Unlike in the earlier studies the majority of the food items contained colourants within the permissible limit. Moreover in samples containing higher concentrations of the colourants, majority of them had only marginal increase from the PFA limit. Many studies were conducted earlier for identification and characterization of colourants used in food items. Ladu, Jilebi and Halwa are the three major sweets of Kozhikode which are being sold in large quantities throughout India. Eventhough no unpermitted colours could be detected in the above food items many of them had colour concentration well above the permitted limit of 100 ppm. The colour combinations used to produce red, brown and green colours had a cumulative concentration of more than double the permissible limit in many samples. It was observed that only two permitted individual colours namely tartrazine (yellow) and sunset yellow (red) were detected in the samples. All other colours were produced by mixing two or more synthetic colourants. In the present study combination of colourants was found to be used for orange (tartrazine and sunset yellow), brown (tartrazine, carmoisine and brilliant blue or sunset yellow, carmoisine and brilliant blue) and green (tartrazine and brilliant blue) colours. All these colourants used in these combinations were from the list of permitted colours by PFA, but the cumulative concentration of these colourants were above the permissible limit in more than 25 % of the samples. 125

8 The major difference observed in the present study from the previous reports are that no unpermitted colours were detected in any of the samples whereas most the previous Indian studied reported excessive use of unpermitted colourants (Tripathy, 2010). On the absence of scientific data the fixing of 100 ppm as the maximum permissible limit was questioned earlier (Pratima and Sudershan, 2008). Apart from the use of higher concentration of the individual colourants this study could not find much variation from the regulations of the PFA Act (2010). In the absence of Acceptable Daily Intake (ADI), fixing the maximum permissible limit at 100 or 200 ppm need re-evaluation to find any adverse reactions. Moreover the use of colour combinations also require further detailed toxicological studies. 126

9 2. GENOTOXICITY OF FOOD COLOURANTS Changing life styles and use of packaged food items has influenced the food consumption pattern of the average Indians (Sridevi and Reddy, 1998). In India majority of food manufacturers are running small scale and cottage industries and hence they are unable to restrict the use of colourants or any other additives as per stipulations of the PFA due to ignorance and/or negligence. This finding is well in agreement with the earlier reports that additives are not used as per the regulations under PFA Act (Pratima and Sudershan, 2008). Azo compounds are the most common synthetic colourings used in the food, pharmaceutical and cosmetic industry. Also known as coal tar dyes, they contain an aromatic ring linked by an azo bond to a second naphthalene or benzene ring. Colouring matter entering the intestinal tract is subjected to the action of acid, digestive enzymes, and microflora. Azo compounds may reach the intestine directly after oral ingestion or through the bile after parenteral administration. They are reduced by azo reductases from intestinal bacteria and, to a lesser extent, by enzymes of the cytosolic and microsomal fractions of the liver (Elizabeth, 1994). Many studies had shown that the food colourants can cause health hazards affecting kidneys and causing allergies, gastrointestinal problems and cancer (Price et al, 1978). The modern technological advancement in food industry has resulted in the use of variety of food colours alone or in combination (Kiple and Ornelas, 2000). Studies have shown that the food colours might cause health hazards affecting kidneys and causing allergies and gastrointestinal cancer. Most 127

10 of the artificial food dyes in use today are of a class of chemicals that act as carcinogens and are banned in some countries (Tsuda et al, 2001; Golka et al, 2004). Additives can induce DNA damage in bacteria, fungi, insects and mammalian cells in vivo and in vitro. They also cause chromosomal aberrations in mammalian cells, including human cells (Hassan, 2010). Food colourants were reported to be toxic to human lymphocytes in vitro and it seems that they bind directly to DNA (Mpountoukas et al, 2010). In the present study an attempt was made to demonstrate the genotoxicity of the permitted food colourants by using the Cytokinesis Block Micronuclei (CBMN) Assay. The study was also extended to evaluate the mitotic division abnormalities in human lymphocytes by varying concentrations below and above the permissible limit of 100 ppm. Tartrazine is one of the most commonly used synthetic colourant all over the world. Several studies have proven that the adverse reactions caused by the use of synthetic colours are not trivial. For example, 0 40% of allergic reactions in asthmatic patients occurs with tartrazine (Kolly et al, 1989). Tartrazine-related hyperactivity in children (Rowe and Rowe, 1994) and induced DNA damage in the colon of mice at doses close to the Acceptable Daily Intake were reported earlier (Sasaki et al, 2002). Tartrazine and sunset yellow was tested for mutagenic activity at a concentration of 0.5 g/100 ml in cultures of Escherichia coli, but no mutagenic effect was observed. The colourants produced side effects like urticaria, rhinitis, nasal congestion, allergy, abdominal cramps and edema (Luck and Rickerl, 1960; Inomata et al, 2006). 128

11 Studies on the effect of tartrazine on the testicular toxicity or on the function of the male reproductive system are scanty. Mangelsdorf et al (2003) indicated that histopathology and reproductive organ weights analysis provide the best means for detecting substances that potentially affect male fertility and that sperm analysis reflects the results obtained by histopathology and measurement of organ weights. Intercellular connections were reduced and imperfect and dilation in some semniferous tubules of testis mice treated 1% tartrazine was observed. Significant damage was observed in testis of mice treated with 2.5% tartrazine; extensive disruption in semniferous tubules, widening of the interstitial spaces and loss leydig cells. Spermatogenic cells are affected and then depleted with absence of spermatozoa in the lumen (Mehedi, et al, 2009). It was observed that tartrazine at the dose of 2.5% in drinking water significantly reduced epididymal and testicular sperm counts including morphological abnormalities. However, the motility was both decreased in 1 and 2.5% treated groups. Tartrazine has toxic effect on the testis and the epididymis of Swiss Albino mice when it is administered at high doses equivalent to mg kg -1 day -1 and it also affects testis structure and sperm motility at middle doses equivalent to mg kg -1 day -1. These doses level were in excess of the ADI of tartrazine (0-7.5 mg kg -1 body weight). No reliable data is available on the daily intake of tartrazine in food. Majority of the food items available in the market contain artificial food additives and tartrazine is present in most foodstuffs especially in drinks and juices (Husain et al, 2006). Sub-chronic ingestion of tartrazine in drinking water can produce adverse effects on fertility, reproductive performance and sperm parameters in male mice at 1 and 2.5% of tartrazine. It 129

12 was proven that above the ADI, tartrazine possess adverse effects. Mehedi et al, (2009) suggested the need for carrying out surveys among the population to estimate their daily intake of additives. Das and Mukherjee (2004) had shown that food colouring agents, amaranth, erythrosine and tartrazine at the concentration of 8mM caused high genotoxicity, cytostaticity and cytotoxicity. The frequency of Sister Chromatid Exchanges/cell was increased 1.7 times over the control level. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these colourants bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. Additionally DNA electrophoretic mobility experiments showed that these colourants are obviously capable for strong binding to linear dsdna causing its degradation. Evaluation of the data and curves by quantitative and qualitative analysis further supported these findings (Tsuda et al, 2001). In the present study it was observed that almost all the food colourants which were isolated and characterized from the samples collected from Kozhikode were found to be toxic to human lymphocytes. The toxicity was observed even at the permissible limit of 100 ppm. Study on reproductive and neurobehavioral toxicity of tartrazine in mice showed that at the dose of 0.45% of tartrazine in the diet produced a few adverse effects in neurobehavioral parameters during the lactation period (Tanaka, 2006). Prolonged use of this colourant increased the number of gastric mucosa lymphocytes and eosinophils of Wistar rats (Moutinho et al, 2007). 130

13 In the present study majority of the samples contained colours which were produced not by a single colourant but a combination of two or more colourants. The colour combinations used to produce orange, brown and green colours had a cumulative concentration of more than double the permissible limit in many samples. The toxicity observed in the present study was proportional to the concentrations of the colourants whether used alone or in combination. Studies on the use of colour combinations are scanty. In the present study tartrazine was found in majority of the food items. In this study it was observed that colour combinations produced comparatively higher genotoxicity than the individual colourants when the total concentration of the combination was the same as that of the individual colourant. The CBMN frequency was reduced drastically by reducing the concentration of the colourants to 50 ppm i.e., 50 percent of the permissible limit of 100 ppm as per the PFA Act of India (PFA, 2010). Sunset Yellow did not induce DNA damage in comet assay or cause frameshift, base pair, or forward mutations; chromosomal aberrations; or cause mitotic gene conversion (Izbirak et al, 1990). Durnev et al (1995) reported that this colourant tested upto 0.8 mg/kg body weight could not induce any chromosomal damage. But it was reported that this colourant can cause systemic toxicity in man (Inomata et al, 2006). Sunset yellow has been shown to cause allergic or intolerance reactions in certain people, particularly those with a pre-existing sensitivity to aspirin, but no mutagenic effect was reported (Health Canada, 2007). In the present study even at the PFA permissible limit, sunset yellow was found to be 131

14 genotoxic. The use of higher concentration was found to increase the risk of toxicity. This is contrarary to the earlier findings. Growth retardation and severe weight loss in animal, cancer, DNA damage, increased tumours in animals were also found in studies dealing with sunset yellow. This additive caused kidney and adrenal tumours in research animals, and has been known to provoke particularly nasty reactions in people, which may include swelling, rashes, stomach pain, and vomiting. It may also cause or worsen ADHD symptoms in children. In addition, this colourant may cause chromosomal damage in animals. It causes side effects like urticaria, rhinitis, nasal congestion, allergy, abdominal cramps & edema (Inomata et al, 2006). In the present study it was observed that this colourants was used above the permissible limit in Ladu, Jilebi and Halwa collected from the local market. Eventhough in the majority of the samples, the colourant used is within the permissible limit, nearly 25 percent samples contain higher concentrations. The genotoxicity was found to be increasing with increasing concentration of the colourant and the toxicity was found to be drastically reduced when the colour concentration was reduced to 50 ppm. No comparison is possible as reports on the genotoxicity of this colourant are scanty in literature. Brilliant Blue was reported to be toxic to the retina. Ueno et al (2007) showed that this colourant cause retinal cell degeneration even at lower concentration than the ADI. But Remy et al (2008) reported no retinal toxicity or adverse effects in animal and human studies. The Panel on Food Additives and Nutrient Sources 132

15 reported that the consumption of this dye was much lower than the ADI and even at the recommended dose of 6mg/kg body weight per day the colourant did not produce any short term or long term toxicity including genotoxicity and carcinogenicity (Lok et al, 2010). In this study a combination of this colourant was found be used extensively in Halwa to produce green colour. Halwa is the only sweet which was available in many exotic colours including green. Green colour can be produced by the combination of brilliant blue and tartrazine. This colourant alone or in combination was found to be genotoxic in the present study, the toxicity being higher when this colourant was used along with other colourants. Carmoisine was reported to be non-toxic and non-carcinogenic by Ford et al (1987), but it undergoes initial reduction followed by the formation of toxic components (Marathe et al, 1993). Toxicological studies of this colourant are scanty and hence the result of these present findings could not be compared with previous reports. In this study this colourant was found to produce genotoxicity in peripheral lymphocyte culture by CBMN assay as in the case of other colourants. The toxicity was found to be increasing with concentration of the colourant. Fast Green FCF is widely used, as food colourant. It is an immunotoxic agent (Reyes et al, 1996). Brown et al (1978) observed that fast green FCF was nonmutagenic in the Salmonella/microsome assay. The bacterial DNA repair tests also showed that the dye is non-mutagenic (Kada et al., 1972; Rosenkranz and Leifer, 1980). 133

16 The usefulness of such primary screening tests combining two different genetic end-points, gene mutation and chromosomal aberration, and some correlation between mutagenicity and carcinogenicity of food additives have been reported by Ishidate et al (1984). Fast green fed to albino rats for 35 days produced harmful changes in the studied blood parameters. These changes could be attributed to possibility for production of carcinogenic nitrosamines within the nitrite-or nitrate-containing foods themselves or in the stomach (Giri et al, 1986). Although all dyes produced mitotic aberrations, fast green FCF showed comparatively stronger clastogenic activity (Roychoudhury and Giri, 1989). Fast green is an immunotoxic agent (Ali and Bashier, 2006). Fast green was shown to be genotoxic in the present study by CBMN assay. This is well in agreement with the previous reports of Ali and Bashier (2006) and Swaroop et al (2011b). The interesting finding in the study is that the toxicity is directly proportional to the concentration of the colourant. Here also the dose of 100 ppm seems to be higher when the genotoxicity is concerned. Ponceau 4R was shown to be mutagenic by Cameron et al (1987). But the mouse micronucleus assay was negative (Hayashi et al, 1988). Cytogenetic study in bone marrow cells showed that the colourant was non-mutagenic (Agarwal et al, 1993). Similarly no evidence of carcinogenicity was reported by the use of this colourant in experimental animals (TemaNord, 2002). In this study eventhough this colourant was not detected in any of the food items, CBMN assay was performed. The results showed that this dye is as toxic as the other colourants which were isolated from the samples. 134

17 Indigo Carmine may affect genetic material and may cause cancer based on animal data. No human data found. Indigo carmine was found to increase the incidence of Sister Chromatid Exchange when tested in mice. Short term in vitro tests with indigo carmine have yielded conflicting results regarding its carcinogenicity, mutagenicity, and genotoxicity. It may cause skin irritation. It may cause respiratory tract irritation. It may cause gastrointestinal tract irritation with nausea, vomiting, and diarrhea (Giri and Mukherjee, 1990). Earlier studies have reported that this food colour can induce clinical symptoms of hyperactivity and Sister Chromatid Exchange (Bateman et al., 2004). In this study indigo carmine was found to be as genotoxic as in the case of other permitted colourants. Sister Chromatid Exchange (SCE) induced by indigo carmine (secondary amine containing dye) may be due to the presence of nitrite singly and or in combination. Erythrosine is used extensively as a colour additive in foods, drugs and cosmetics. At 300 µg/ml, erythrosine produced an increase in micronucleus frequency in the absence of hepatocytes. A dose related increase in the mitotic frequency was observed due to an increase in the number of first mitosis. Thus increased genotoxicity was observed only at concentrations well in the range of cytotoxicity (Rogers et al, 1988). The WHO/FAO recommends an Acceptable Daily Intake of 0.1 ppm for this additive (Roche, 1989). Erythrosine at lower concentrations showed stimulatory effect on mitotic index, while at higher concentrations showed inhibitory effect on mitotic index. Both erythrosine and brilliant blue induced several chromosomal aberrations like 135

18 breakage, stickiness, chromatid separation, scattering, extrusion, chromosomal bridge, polarity abolition etc. It was also proved that at higher concentrations these colourants were cytotoxic and mutagenic to the chromosomes of Allium sativum in the form of lethal aberrations and these concentrations were also responsible for inhibiting the mitotic activity significantly (Yadav et al, 2005). Vivekanandhi (2006) showed dose-dependent decrease in cell proliferation in Swiss albino rats. In the present study a significant increase in the CBMN frequency with increased dose of erythrosine exposure was observed which is in agreement with the previous report. Erythrosine was reported to cause chromosomal structural aberrations which may be suggestive of its mutagenicity (Hamdy, 2000). In this study it was observed that this colourant is toxic even at the permissible limit of 100 ppm and the toxicity was reduced only when the dose was reduced to 50 ppm i. e half the permissible limit. Single higher dosage ( mg/kg, p.o) of erythrosine administration to young adult male rats reduced motor activity by reducing serotonergic activity with modulation of central dopaminergic activity depending on the brain regions (Dalal and Poddar, 2009). Synthetic colourants are extensively used around the world in processed food and beverages. The list of permitted synthetic colourants varies from country to country. The list of US FDA has included seven colourants whereas in India the list contains eight and in Japan it is twelve. The mutagenicity of 4 azo dyes namely amaranth, allura red, new coccine, and tartrazine were reported to induced DNA damage in the colon at close to the Acceptable Daily Intakes (ADIs) (Sasaki 136

19 et al, 2002). Of the above synthetic colourant only tartrazine is included in the list of permitted colourant in India. The individual response of each varies not only according to dose, age, gender, nutritional status and genetic factors, but also according to long term exposure to low doses (Sasaki et al., 2002). Adverse hyperactive behavioural changes in children have been documented with excess amounts of artificial food colourings and sodium benzoate preservatives in the diet (Bateman, 2004). The genotoxicity of food colour additives are dependent on their conversion to reactive metabolites, the activation is accomplished by acetyltransferases, which are widely distributed in animals. For carmoisine, tartrazine and ponceau 4R, saturation of ADI ranged from 27.4% to 90.3% in children and adolescents and from 10.8% to 47.6% in adult subjects. These results indicate that children and adolescents are more vulnerable to higher intakes of food colours compared with the adult population (Dixit et al, 1995). In this study food colourants were tested at varying concentration ranging from ppm. This is the first attempt to correlate the genotoxicity with the concentration of the colourants. At concentration of 100 ppm and above food colourants caused genotoxicity and the toxicity was proportional to the concentration of the colourants used. In this study the genotoxicity of all the eight colourants permitted under FDA as well as their combinations were evaluated using CBMN assay. All the colourants alone or in combinations were found to be genotoxic, but the toxicity varied from colourant to colourant and also from combination to combination. 137

20 The maximum toxicity was observed when a combination of sunset yellow and brilliant blue was used. In the case of individual colourants the maximum toxicity was for indigo carmine followed closely by carmoisine and ponceau 4R. But we have not detected any of the above colourants in the materials collected for the study. Tartrazine, the colourant use of which is maximum in India and in other countries is the least toxic of all, if used alone. If it was used in combination with other colourants the toxicity was found to be increasing even if the total concentration of the colours in the combination was within the permissible limit of 100 ppm. The ADI for permitted synthetic colours varies from 0.1 to 25 mg per kg bodyweight, i.e. a difference of 250 times in developed countries (CAC, 1999). The rationale of fixing a uniform regulatory limit of 100 ppm to the permitted colours in all foods by the PFA is not justified. There is a need to develop regulatory limits for additives based on risk assessment taking into consideration of food habits and technological necessity. 138