1
A study of the effects of Allura Red AC (Red 40) and Tartrazine (Yellow 5) individually
and in combination, on development of Xenopus laevis (African clawed frog).
By
Brittanie L. Fils
Spring 2019
A thesis
Submitted in partial fulfillment
Of the requirements
For a baccalaureate degree
In Biology
In cursu honorum
Reviewed and approved by:
______Dr. Laura Twersky Thesis Supervisor
Submitted to The Honors Program, Saint Peter’s University
2
Acknowledgements
I would like to thank my family, especially my older brother, Steven for being part of an amazing support system. I would also like to thank my close friends including but not limited to,
Kayla Murray, Jack Payne, Sanket Kumar, and Naomi Dawkins. Most of all, I would like to thank Dr. Laura Twersky for her patience, understanding, and advice throughout the years and work on this project.
Finally I would like to thank the TriBeta Research Foundation for supporting this project through a grant. 3
Table of Contents
Cover Page ...... 1
Acknowledgments ...... 2
Table of Contents ...... 3
Abstract ...... 4
Introduction...... 5-12
Materials and Methods ...... 12-15
Results ...... 15-18
Discussion ...... 18-19
References ...... 20-23 4
ABSTRACT
The consumption of artificial food dyes is a topic of controversy in the United States. In contrast, many European nations opted for natural alternatives after studies suggested that they cause hypersensitivity in children. Two of the most prevalent food dyes, Red 40 and
Yellow 5, are heavily debated as carcinogens and are solidly linked to ADHD in children.
The purpose of this study is to use the model organism, Xenopus laveis, the African clawed frog, to observe the effects of these dyes separately and together. Specimens will be incubated in different concentrations of Red 40, Yellow 5, or both at either 2.5μg/ml or
5μg/ml. There will be one control group and eight experimental groups. These groups will be exposed to the dyes from metamorphosis stage 48 (Nieuwkoop and Faber) until adulthood. They were observed by determining how long movement lasted once it began.
The results suggest a strong link to hyperactivity and possible effects on development, such as different mortalities among the groups. Tadpole length was in terms of length, and any malformations were documented. A review was also conducted on dyes present in common products available to consumers, which will be discussed.
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INTRODUCTION
Food dyes are used by large corporations to create a uniform and appealing product to the masses. These synthetic dyes have been used by modern, Western nations for at least a hundred years. Dyes are organic (carbon-containing) chemicals. These dyes were originated from coal tar, and are now made from petroleum (Kobylewski & Jacobson, 2012). In the United
States, since 1955, the consumption of food dyes has increased five times over. Only three of these dyes, Allura Red AC (Red 40), Yellow 5 (Tartrazine), and Yellow 6 (Sunset Yellow FCF) make up ninety percent used in foods. These additives are not necessary and only serve aesthetic purposes. In recent times, there have been evidence suggesting food dyes have carcinogenic, neurological and hypersensitivity effects, but more specifically, there is evidence suggesting that they causes both hypersensitivity and attention deficit hyperactivity disorder (ADHD) (Bateman et al., 2004).
Xenopus laevis is being used as a model organism for a few reasons. For one, during the tadpole stage, the organism is transparent making it ideal for this experiment. It allows observation and documentation of the specimen. The adverse effects will be documented through all the stages, as well as mortality. This assessment will show us which dye is most likely to cause cancer. Thus, Xenopus laevis, as a model organism, will show which dye, Yellow 5 or Red
40, most likely causes cancer in pre-pubescent children. That would allow any possible carcinogenic effects of Red 40 and/or Yellow 5 to be documented and observed.
Tartrazine or Yellow 5 is an orange-colored water soluble powder (Tanaka et al., 2008).
A search through the common American household shows that it is used in products ranging from sodas to shampoos. The prevalence of this color is troubling considering the fact that it causes allergic reactions. Like Red 40, Yellow 5 is associated with hyperactivity with children 6
and it causes DNA damage in the colons of mice. Damage occurs at close to the acceptable intake value (Rowe and Rowe, 1994). Yellow 5 also causes other changes such as restlessness and issues with sleep (Tanaka, 2006). Interestingly, other food dyes do not cause damage in the colon in a statistically significant manner (Poul et al., 2009). All of this information makes
Yellow 5 pertinent to this study and further studies of food dyes.
ADHD is a childhood-onset neurodevelopmental disorder that affects about 1.4-3.0 percent of children. It is more common in males. ADHD is primarily inherited, but other factors such as exposure to lead play into it as well. ADHD is treatable, but will most likely lead to other mental and social issues later on in life. For that reason, if there is a way to cut down on the number of children suffering from it, steps should be taken. (Thapar and Cooper, 2015). The diagnosis of ADHD requires the symptoms to be present in a variety of settings. The symptoms must be debilitating in some way including but not limited to academics or social situations. The age of diagnosis must be made before the age of twelve according to the American Psychiatric
Association's Diagnostic and Statistical Manual of Mental Disorders 5th edition (Thapar and
Cooper, 2016). The unnecessary increase led to concerted efforts to curb the use of food dyes.
While the association of food additives with hyperactivity is accepted, there is no conclusive evidence to prove that these additives have a negative effect on human development (Lau et al.,
2005).
In 2008, the Center for Science in the Public Interest (CSPI) petitioned the Food and
Drug Administration to ban artificial food dyes. Food dyes are used heavily in foods marketed and consumed by children. Examples are breakfast cereal, candy, and vitamins. Even when parents feed their children fresh fruit, such as oranges, there is a strong possibility that food dyes were used to enhance its physical appearance. The most common way to ingest dyes is through 7
beverages. (Batad and Jacobson, 2016). The CPSI were successful in influencing the United
States government to ban Violet 1 in 1973 because it was linked to cancer in animals. On the other side of the argument, the Internal Association of Color Manufacturers (a trade association for food dye makers and users) claim that food dyes correct natural colors and give an identity to food through color. It is also used to give color to food that have been stored long term. In terms of vitamins, the argument is that the food dye colors allow for an easy, efficient way to identify the pills (Potera, 2010). However, it should be noted that these dyes are possibly used because they are less expensive, easier to preserve and are able to make brighter colors (Kobylewski &
Jacobson, 2012).
There are currently nine FDA approved dyes. Besides Allura Red AC, Tartrazine, and
Yellow 6, the other dyes include, Brilliant Blue (Blue 1), Indigo Carmine (Blue 2), Citrus Red 2,
Green 3, Orange B, Erythrosine (Red 3). There are several banned dyes such as Green 1 which was found to cause liver cancer in animal studies, and Red 2 which is a possible carcinogen
(Kobylewski & Jacobson, 2012). In terms of United States, there are a few, albeit, antiquated laws concerning dyes. Before 1960, the law required that dyes could not be used at all if there were not completely harmless. However, that standard is impossible to live up to. In 1960,
Congress passed the 1960 Color Additives Amendment that simultaneously lessened the standards of acceptable dyes and made the regulations more clear. The law included provisions including testing each batch of food to test if it was an acceptable level of contamination.
Preservatives and flavorings are not tested. Due to a loophole, companies are allowed to label their products as “generally safe for use” even if it has not been subject to toxicity testing
(Kobylewski & Jacobson, 2012). The Federal Drug Association has legal limits for carcinogens in dyes. Within a lifetime of consumption, these dyes can’t result in cancer in one person out of a 8
million. However, these standards are based on consumption rates of Americans in 1990, but the consumption of food dyes has increased fifty-percent since then (Kobylewski & Jacobson, 2012).
Sodium benzoate is a common additive that acts as a preservative in popular drinks that contain food dyes. Like the dyes themselves, sodium benzoate have been linked to ADHD.
Sodium benzoate is the sodium salt of benzoic acid. There have been studies showing that sodium benzoate had a negative effect on dopamine neurotransmission in zebrafish. Exposure to sodium benzoate causes neurotoxic effects in developing larvae. This change had an adverse effect on the survival rates of zebrafish embryos. This change is important because ADHD symptoms are often associated with the dopamine transporter and stimulant medications that change dopamine are helpful in the treatment of ADHD (Beezhold et al., 2012).
The European Food Safety Authority (EFSA) detailed permitted levels of any and all dyes in 2009 in terms of milligrams per kilogram.
TABLE 1. Permitted levels of dyes in food by the European Safety Authority
European Food Safety Authority [EFSA] (2009).
Foodstuffs Maximum permitted level (mg/kg)
Luncheon meat 25
Soups 50
Processed cheese 100
Smoked fish 100
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In the United States, there are few laws, the Federal Drug Association (FDA) has policies regarding fruit and the use of artificial dyes. Specifically, “Artificially colored oranges received in bulk and sold at a retail establishment are not required to bear a label statement declaring the use of artificial coloring…” (U.S Food & Drug Administration).
Previous Study on Food Dye Prevalence and Marketing Towards Children
A recent study detailed the prevalence and frequency of dyes in the United States, particularly, in North Carolina. The most commonly used dye was Red 40 with about thirty percent of sampled products containing it. Yellow 5 was contained in about twenty-one percent.
When it comes to different products, candy, fruit snacks, and drink mixes/powders had a high percentage of dyes. These are the products targeted towards children. The marketing included in some form one of the following: use of a cartoon character, a child-licensed character on the front of the package, a prize incentive and/or bright coloring.
Table Two is a concise summary of findings of dyes from Stevens et al. (2014). The table details the percentage of items sampled that contained Allura Red AC and/or Tartrazine. The findings were found using a Power Wave X spectrophotometer. There are values taken for each sample and then each serving size recommended. (Batada and Jacobsen, 2016).
TABLE 2.
Percentage of food items that contain Allura Red AC and/or Tartrazine.
Stevens et al. (2014)
Product Category Number of Allura Red AC Tartrazine
Products Sampled Percentage Percentage
Candies 27 77.8 63 10
Product Category Number of Allura Red AC Tartrazine
Products Sampled Percentage Percentage
Toiletries 47 22.5 6.4
Condiments 25 28 12
Dairy Products 50 20 8
Cereals 104 25 17.3
Drink 39 71.8 30.8
mixes/powders
Juice drinks 95 15.8 7.4
Canned/packaged 79 6.7 26.9
pastas/soups
Produce 6 0 0
(lettuce, etc.)
EXTRACTION METHODS OF DYES FROM PRODUCTS
There is a strong need for further studies and ways to extract food dyes from their products. Although the Federal Drug Association requires a list of ingredients to be listed on products, the amount of the product is not known. There are a few known methods to discover the amount. 11
A. High-Performance Liquid Chromatography (HPLC)
HPLC is used on potato chips, biscuits, drinks, desserts, sweets/candies, ice cream, etc.
The extraction procedure differs if the sample is a solid or a liquid. Both samples are 5
grams. The solids are vortexed, sonicated, and are placed into a centrifuge for five
minutes at 2500 rpm. For liquids, five grams are dissolved and centrifuged for ten
minutes at 13,000 rpm. The clear top supernatant (50 mL) from the centrifuged liquid
was removed (Lok et al., 2010).
B. UV-visible spectrophotometer
A UV-visible spectrophotometer is used to analyze granulated drinks such as soft drinks.
A two gram sample is dissolved in fifty milliliters of distilled water. Then it is stirred for
five minutes with a magnetic stirrer and the solution is diluted with a solution that is
three parts 0.1M HCL and ten parts 0.1 M NaOH separately (Turak and Ozgur, 2013).
C. Capillary Electrophoresis
Capillary electrophoresis is used to extract dyes in beverages. A 25 mM sodium
phosphate buffer and 25 mM sodium borate buffer (1:1) (pH 8.0) containing 10 mM
sodium dodecyl sulfate (SDS) is used as the extraction method (Suzuki et al. 1994).
NEED FOR EXPERIMENTATION
There is a need for further research since recent studies conflict with each other, and these dyes are prohibited in Europe, but not in the United States. There have been other studies that involve dyes in combination with other substances such as artificial sweeteners including aspartame. The study showed there is a negative synergic reaction that adversely affects the development of the neurological system (Lau et al., 2006). However, another study concluded
Red 40 did not cause cancer in mice. Something notable about this study is that the maximum 12
concentration was not used. The carcinogen in all three of these dyes is benzidine. It is linked to bladder cancer. Usually it is safe for consumption in lower levels. Interestingly, studies have a short period; Studies end in two years or less. Cancer in rodents would show up in the third year
(Potera, 2010). Another study focused on the diet of children. The children’s diet was examined to see if there is a correlation between food dyes and their behavior. In terms of their results, once there was a withdrawal, the hyperactivity decreased. Based on these two studies, there is a need to further examine what effects food dyes have (McCann, 2007).
In this experiment, a concentration of either 5μg/ml or 10μg/ml was used for a trial run.
Also, developmental issues were looked at as well. The primary reason for the experiment is to watch Xenopus laevis’ activity and to monitor its growth, and see if it has abnormal or increased activity as opposed to the control group. Based on all the literature reviewed, this study is unique because it involves a combination of two dyes along with a separate analysis of the dyes. This will give a new perspective on the topic of food dyes because consumption of food dyes is never completely isolated. It is often consumed in conjunction with other dyes. This study will be able to get closer to the way we normally inject food dyes. The large number of groups help determine if one dye affects hyperactivity more than another, and if that effect is changed when the dyes are used together. Pictures were taken at each stage of development to see if the dyes caused any other secondary effects.
MATERIALS AND METHODS
A. Animal Care & Maintenance
The model organism used is Xenopus laevis (clawed frog). They were purchased from
Xenopus 1 and were studied throughout their tadpole stages. Tadpoles are housed in aged (24 hour) tap water. The tadpoles are fed when they hatch out, they are fed Fleischmann's ActiveDry 13
Yeast Original that is also diluted in aged tap water. The Red 40 and Yellow 5 were introduced both together and separately at different concentrations. There was be one control group and 8 experimental groups. Animal development was observed and documented using the stereomicroscope Unitron Z850; during their development they were kept and observed in finger bowls.
Every week a large amount of water is lost due to evaporation. When the water levels are too low then the tadpoles would suffocate. To prevent this from happening all finger bowls were at a minimum volume of 100 ml at all times. The tadpoles also were fed two to three times a week based on water opacity. The yeast was dissolved in water first to prevent the tadpoles from choking.
The solutions used were Allura Red 40 at 2.5μg/ml and 5μg/ml. Yellow 5 at 2.5μg/ml and
5μg/ml. Both dyes together at 2.5μg/ml and 5μg/ml. Both dyes with Red 40 at 5μg/ml and
Yellow 5 at 2.5μg/ml. Both dyes with Yellow 5 at 5μg/ml and Red 5 at 2.5µg/ml. Each of these solutions were made at a 100ml volume. The tadpoles were fed Mondays, Wednesdays, and
Fridays. During these times of feeding the tadpoles were observed, the water and solutions were filled as need. Any malformation developments of the tadpoles were photographed. Any death was photographed.
B. Solution Preparation
Both of the dyes were purchased from Sigma Aldrich. All solutions were made in aged tap water. The dyes come into a powder form, and were weighed out on an analytical balance that was accurate to 0.1 milligrams.
C. Incubation in Dyes 14
All of the groups were incubated at the same stage of their life cycle; metamorphosis, stage 48. (Nieuwkoop and Faber stages). Stage 48 was because of convenience. The dyes arrived at that this stage. Xenopus laevis was kept at room temperature throughout the experiment. The study was continued throughout their tadpole life cycle. The groups of tadpoles were given their respective concentrations of both Red 40 and Yellow 5 throughout the experiment and into maturation. Each group had a stock solution ready at all times to account for evaporation. The stock solutions are kept in flasks that are capped. The control group had aged tap water as its own stock. This tap water is also used to dilute the Fleischmann's Yeast. In case of emergency, dangerously low water levels, the aged tap water is to be used in place of stock solutions.
Table 3.
Concentration of Allura Red AC and Tartrazine Solutions
Group Number Allura Red AC Concentration Tartrazine Concentration
Control None None
Group #1 2.5μg/ml None
Group #2 5.0μg/ml None
Group #3 None 2.5μg/ml
Group #4 None 5.0μg/ml
Group #5 2.5μg/ml 2.5μg/ml
Group #6 5.0μg/ml 5.0μg/ml 15
Group Number Allura Red AC Concentration Tartrazine Concentration
Group #7 5.0μg/ml 2.5μg/ml
Group #8 2.5μg/ml 5.0μg/ml
D. Measuring Activity
The way that hyperactivity was recorded is by movement. Each tadpole was be evaluated and the average was be taken for the group in seconds. Movement is defined here as any motion the tadpole makes from start to stop. The movement was measured by how long it took for movement to stop. The tail is the primary and standard gauge for movement used in this experiment. Distance moved was not measured.
E. Photography
Animal development was observed and documented using the stereomicroscope Unitron
Z850. During their development they were kept and observed in finger bowls. It should also be noted that any malformation in the tadpoles were noted during development. Deformity may affect how the tadpoles move.
RESULTS
In terms of movement, the highest amounts of Allura Red AC (RAC) by itself yielded the highest amounts of observed movement. The second highest level of movement came from equal levels of 2.5 μg/ml. Lower levels of movement came from the combined higher level of
Tartrazine (T) and lower level of Allura Red AC. 16
The control had an average length of 2.5 centimeters. Group one (2.5 μg/ml RAC) had an
average length of 1.9 centimeters. Group three (2.5 μg/ml T) had an average length of 1.8
centimeters. Group four (5.0 μg/ml) had an average length of 1.3 centimeters. Group five (2.5
μg/ml RAC, 2.5 μg/ml T) had an average length of 2.2 centimeters. Group six (5.0 μg/ml RAC,
5.0 μg/ml T) had an average length of 1.2 centimeters. Group seven (5.0 μg/ml RAC, 2.5 μg/ml
T) had an average length of 2.2 centimeters. Group eight (2.5 μg/ml RAC, 5.0 μg/ml T) had an
average length of 1.8 centimeters.
On March 19th, 2018 the incubation in solution started. Seven days later, two from group
4 died and it was unclear how it died. Sixteen days after incubation, group 8, one of the tadpoles
had died, group 2 (5.0 μg/ml RAC) had two dead tadpoles. Twenty-one days after incubation,
group 2 had another death leaving only one left in the group. Fifty days later, one from group 8
died. It was examined under a microscope. One small white growth was present. In all these
instances, the reason for the mortality was inconclusive.
Due to the cost of Red 40 and how little of it can be obtained, on March 28th the solution
was diluted for about a week. The concentration was about ten percent weaker. Thirty-two days
after incubation, movements were recorded for length of time, and overall size were recorded.
Movement was measured when it was unprompted, meaning there was stimulus to induce
movement. It was then measured to observe how long this movement lasted in seconds.
Table 3.
Movement in seconds
Control G1 G2 G3 G4 G5 G6 G7 G8
Movement 1s, 3s 7.5s 66s 23s 5s 46s 27s 11s 3s
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Table 4.
Length
Control G1 G2 G3 G4 G5 G6 G7 G8
Length 2.4cm 1.9cm 2.9cm 1.7cm 1.3cm 2.4cm 0.9cm 2.5cm 1.8cm (cm) 2.3cm 1.2cm 1.9cm 1.2cm 2.2cm 1.2cm 2.2cm 1.9cm 2.7cm 2.5cm 1.5cm 1.5cm 1.7cm 1.4cm 1.8cm 1.6cm 2.2cm 2.4cm 1.3cm
Photography
Figure 1.
This picture was taken on April 23rd, 2018 and shows one of the dead tadpoles from Group 7.
There appears to be a small, abnormal growth present. 18
Figure 2.
This picture was taken after fifty days of incubation and shows one of the tadpoles from group 8 after it died. There is a small growth present.
DISCUSSION
The health of each of the groups were checked three times a week. Group 2 (5.0 RAC) had the highest mortality rate and also had the fastest growth. All tadpoles started at the same stage but experimental group 2 consistently reached other developmental stages first and had more movement when recorded and even when observed when the tadpoles were being fed. The 19
groups that contain yellow dye had consistently smaller tadpoles. In terms of a possible synergistic reaction, group 6 contained equal amount of red and yellow dye at 5μg/ml. They were the smallest group. Group 8 had more red than yellow and had the lowest movement. As of now, it seems like red may the effect of making the tadpoles larger while, yellow in combination with red only at equal concentrations makes them much smaller than the control group. It is important to note that these are preliminary results. There is a need to investigate this topic with larger sample sizes and repetition of the study.
Food dyes continues to be used more and more commonly in products within the United
States. Children are more vulnerable to the use of dyes because they are still developing and many of those products are marketed towards children. There are few studies that focus on food dyes and product manufacturers are not required to release or inform consumers of the amount of dyes that their products contain. There is a significant lack of studies concerning food dyes and other environmental factors. There are no studies that focus on dyes and the microbiome, environmental endocrine disruptors, sugars, artificial sugars, and other dyes in combination with each other. There is also the issue of cancer. The clawed frogs’ cleavage stages are a model system analogous to rapid cell division seen in tumors. Since cancer is linked to both of these dyes it should be researched as well.
20
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Addendum
Nieukoop and Faber stages
Nieuwkoop and Faber (1994).
Nieuwkoop and Faber 1994. Normal Table of Xenopus laevis (Daudin). Garland Publishing Inc,
New York