Effects of maternally exposed colouring food additives on cognitive performance in rats

http://tih.sagepub.com/Toxicology and Industrial Health

http://tih.sagepub.com/content/early/2012/02/09/0748233712436638The online version of this article can be found at:

DOI: 10.1177/0748233712436638

published online 9 February 2012Toxicol Ind HealthDuygu Kumbul Doguc, Betul Mermi Ceyhan, Mustafa Ozturk and Fatih Gultekin

Effects of maternally exposed colouring food additives on cognitive performance in rats

Published by:

http://www.sagepublications.com

can be found at:Toxicology and Industrial HealthAdditional services and information for

http://tih.sagepub.com/cgi/alertsEmail Alerts:

http://tih.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- Feb 9, 2012OnlineFirst Version of Record >>

at Süleyman Demirel Üniversitesi on March 4, 2012tih.sagepub.comDownloaded from

http://tih.sagepub.com/

http://tih.sagepub.com/content/early/2012/02/09/0748233712436638

http://www.sagepublications.com

http://tih.sagepub.com/cgi/alerts

http://tih.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://tih.sagepub.com/content/early/2012/02/09/0748233712436638.full.pdf

http://online.sagepub.com/site/sphelp/vorhelp.xhtml


Effects of maternally exposedcolouring food additives oncognitive performance in rats

Duygu Kumbul Doguc1, Betul Mermi Ceyhan1,Mustafa Ozturk2 and Fatih Gultekin1

AbstractArtificial food colourings and additives (AFCAs) have long been suggested to adversely affect the learning andbehaviour in children. In this study, we aimed to provide additional data to clarify the possible side effects ofcolouring additives on behaviour and memory. We administered acceptable daily intake values of AFCAs as amixture (Eritrosin, Ponceau 4R, Allura Red AC, Sunset Yellow FCF, Tartrazin, Amaranth, Brilliant Blue,Azorubin and Indigotin) to female rats before and during gestation and then tested their effects on behaviourand on spatial working memory in their offspring. Effects on spatial learning and memory were evaluated byMorris water maze, behavioural effects were evaluated by open-field test and forced swim test. Our resultsshowed that commonly used artificial food colourings have no adverse effects on spatial working memory anddid not create a depressive behaviour in offspring. But they showed a few significant effects on locomotoractivity as AFCAs increased some parameters of locomotor activity.

KeywordsArtificial food colourings, Morris water maze, spatial learning, open field test, forced swim test

Introduction

Artificial food colourings, additives (AFCAs) and

some preservatives have long been suggested to

adversely affect the learning and behaviour in

children. The suspicion of main putative effect of

AFCA is to induce overactive, impulsive and inatten-

tive behaviour (i.e. hyperactivity) in children which

should be associated with attention-deficit/hyperac-

tivity disorder (ADHD) (Aguiar et al., 2010; McCann

et al., 2007).

Ben Feingold made his initial claims of the detri-

mental effect of AFCAs on childhood behaviour more

than 30 years ago. Feingold (1976) hypothesized that

hyperactivity might be a child’s adverse reaction to

food additives, such as artificial sweeteners, artificial

colours and preservatives, that are present in

numerous industrial foods and drinks (Bellisle,

2004; Feingold, 1976). Since then different kinds of

AFCAs were studied separately or as a mixture in

children with ADHD or in experimental studies.

Tanaka studied the AFCAs separately in experimental

studies in rats and focused on reproductive and

neurobehavioural effects on them. In most of these

experimental studies, neurobehavioural procedures

evaluated especially the neuromuscular developmental

effects of AFCAs (Tanaka, 1993, 1994, 1996, 1997,

2001, 2006).

Disputes about this case caused concerns about the

safety of AFCAs in the consumers’ minds. European

Food Safe Authority (EFSA) took these concerns into

consideration and therefore, some of the colouring

additives were decided to be reevaluated. At the end

of these reevaluations, acceptable daily intake (ADI)

values of some of the colouring additives were

1Medical Biochemistry Department, Medical Faculty of SuleymanDemirel University, Turkey2Public Health Department, Medical Faculty of Suleyman DemirelUniversity, Turkey

Corresponding author:Duygu Kumbul Doguc, Medical Biochemistry Department,Medical Faculty of Suleyman Demirel University, Turan Mah2213 Sk. Halıcı Evleri B Blok Kat:4 Daire:9, 32000 Isparta, TurkeyEmail: [email protected]

Toxicology and Industrial Health1–8© The Author(s) 2012Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0748233712436638tih.sagepub.com



decreased. However, studies on the effects of AFCA

on memory and neurobehaviour is still inadequate in

healthy individuals. In this study, we aimed to provide

additional data to clarify the possible side effects of

colouring additives on behaviour and memory. For

this purpose, we administered a mixture of AFCA

which is commonly present in many industrial foods

to female rats before and during gestation and then

tested their effects on behaviour and spatial working

memory in their offspring when they became adult.

Materials and methods

Materials

The additives in the artificial food colours mixture and

the doses used in the study were as follows: Eritrosin

0.1 mg/kg/day, Ponceau 4R 4 mg/kg/day, Allura Red

AC 7 mg/kg/day, Sunset Yellow FCF 2.5 mg/kg/day,

Tartrazin 7.5 mg/kg/day, Amaranth 0.5 mg/kg/day,

Brilliant Blue FCF 12.5 mg/kg/day, Azorubin 4

mg/kg/day and Indigotin 5 mg/kg/day. The doses

used in the study were the ADI values of these artificial

food colours which were obtained from Joint Expert

Committee on Food Additives (JECFA) monographs

(http://www.inchem.org/pages/jecfa.html). To prepare

the artificial food colour mixture, colour additives were

weighed separately and dissolved in water. Artificial

food colour mixtures were prepared weekly and stored

at 4�C.

Animals

Thirty, Wistar Han strain, female rats were included

in the study and categorized as control group

(n ¼ 15) and experiment group (n ¼ 15). The rats

were housed individually in solid-floored cages with

wood flakes and kept in a temperature-controlled

(23 + 1�C) room. One millilitre of artificial food col-

our mixture and 1 ml of water have been administered

daily for a week by oral gavage to the experiment and

the control groups, respectively. At the end of admin-

istration week, one male rat was placed in each cage

and was allowed for 5 days to stay at the same cage

to mate and the pregnancies of rats were provided.

During the pairing period and the pregnancy period,

administration of artificial food colour mixture to

experiment group and water to control group was con-

tinued with the same dose and in the same way. After

the partus of pregnant rats, offspring were allowed to

stay with their mothers for a month for breast-feeding.

Afterwards male and female offspring were separated

and offspring of artificial food colour mixture

administered rats were accepted as experiment group

and offspring of water administered group were

accepted as control group. At the end of 3 months,

10 female and 10 male rats from experiment group

and 10 female and 10 male juvenile rats from control

group were selected. The rats were grouped as female

experiment group (FEG, n ¼ 10), female control

group (FCG, n ¼ 10), male experiment group (MEG,

n ¼ 10), and male control group (MCG, n ¼ 10). The

colony room was maintained under an automatically

regulated 12:12-h reverse light/dark cycle (lights off

at 08:00 a.m.) and testing occurred during the dark

phase which is their most active period.

For the spatial working memory, anxiety and

depression tests, the rats were transferred to the

laboratory where the test would be done and waited

for a week for adaptation.

The animals were handled under the prescriptions

for animal care and experimentation of the pertinent

European Communities Council Directive (86/609/

EEC), and all the procedures were approved by the

Ethical Committee of Suleyman Demirel University.

Behavioural tests

Morris water maze. Morris water maze (MWM) is a

task that evaluates spatial learning, a measure of cog-

nitive function. A virtual version of this test has been

validated in the human as a test for spatial learning.

The tank was placed in a dimly lit, soundproof test

room with various visual cues. This task uses a round

pool of water in which a platform is submerged

beneath the surface. When placed in the maze the ani-

mal’s task is to find the hidden platform. The MWM

consisted of a circular pool, 150 cm in diameter and

80 cm in height, with the interior painted white. The

water was maintained at a temperature of 22 + 2�Cand was made opaque by the addition of nontoxic

dark yellow paint. The pool was surrounded by four

halogen lights which were directed to the walls that

surrounded the room.

Experimental procedure. Each trial was tracked

using an overhead camera interfaced with a computer

that recorded the time and path travelled for all the

runs in the water maze. The tracking program which

we used was Smart Version 2.0. The maze was

divided into four equally sized virtual quadrants,

which was named as zone 1, zone 2, zone 3 and zone

4. The platform remained in a fixed location for all

2 Toxicology and Industrial Health



runs and the target quadrant was the fourth quadrant.

Throughout the experiment, animals were handled

before the first trial of each day and then were

released once from each of the four quadrants facing

the centre of the pool. Daily training consisted of four

trials in which the rat was placed in the water from

four random starting positions (1, 2, 3 and 4) and the

latency of escaping onto the platform was recorded.

Start locations were equally spaced around the peri-

meter of the pool. This was conducted for 5 consecu-

tive days. In a protocol modified from Morris (1984),

acquisition of place learning using spatial cues and

navigational strategy was done on days 1–5 and then

the test for memory called ‘probe trial’ was performed

on day 6. On the first day of testing, animals were

allowed to swim in the pool for 90 s. If a rat could not

find the hidden platform, it was placed on the plat-

form for 30 s to introduce the platform and show that

the platform is a mean of escape from the water. On

days 2–5, the animals were allowed to swim in the

pool for 60 s. A trial ended when the rat climbed on

the platform. If a rat again had not found the platform

after 60 s, it was placed on the platform by the experi-

menter. All the rats were left on the platform for 15 s

and then removed to their home cages by the experi-

menter. There were four trials per day, with an inter-

trial interval of approximately 20 min for each rat.

After trials, subjects were dried with a towel and

warmed under a 40-W soft white bulb (OSRAM)

before being returned to the home cages. At the end

of the fifth day, each rat had been trained 20 times

totally and the acquisition period had been completed.

On day 6, the platform was removed and the rats were

released from the three other quadrants which did not

contain the hidden platform before. At this probe trial

test, the time spent in the target quadrant where the

platform had been during acquisition period was

recorded. The percentage of swimming in the quad-

rant of the former platform was calculated as a mea-

surement of spatial memory. At day 1 if the time

taken was greater than 90 s, it was recorded as 90 s;

since day 2 if the time taken was greater than 60 s,

it was recorded as 60 s. On day 7, visible platform

procedure was applied. This is the cued version of the

Morris water escape task. To eliminate the direct or

indirect effects of the drug we have used, such as

decreased vision, decreased motivational factors,

increased anxiety of animals in the water and there-

fore decreased the desire to escape, we used cued ver-

sion procedure. Each rat was released from the fourth

quadrant all the time and visible platform was carried

to the different quadrants for each trial except the

fourth quadrant. Again escape latency to the visible

platform was recorded (Morris, 1984).

Open-field test. The open-field maze (arena) was used

to study locomotor, exploratory and anxiety-related

behaviours. The apparatus consisted of a spherical

arena (150 � 150 cm). The floor was marked into

16 segments. Four halogen lights were directed to the

walls surrounding the apparatus, so diffuse overhead

illuminations were provided. The animals were tested

in a quiet room and the locomotor activity over a

5-min period was recorded using a ceiling-mounted

videocamera (Sony SSC-DC398P, Japan). Each ani-

mal was tested individually in the open-field maze.

Each animal was allowed to explore the maze for

5 min and behaviours were scored. The behaviours

scored were as follows: the number of lines crossed with

at least three paws of the rat, number of rearing and num-

ber of walling, the time spent at the edge of the appara-

tus, the time spent at the centre of the apparatus and

number of centre square activity was recorded by a rater

who were blind to the animals’ housing conditions. Line

crossing (the number of line crosses) is a form of loco-

motor behaviour—horizontal locomotor activity. Rear-

ing is defined as the animal standing upright on its hind

legs. Walling is defined as the animal standing upright

on its hind legs and front legs on the maze’s walls. Line

crosses, rearing, walling, central square frequency and

central square duration are usually used as measures

of locomotor activity as well as exploration and anxiety,

with a higher frequency of these behaviours indicating

increased locomotion and exploration and low anxiety.

Edge duration is a measure of exploratory behaviour

and anxiety with a higher frequency of these activities

indicating lower exploratory behaviour and higher anxi-

ety (Brotto et al., 2000; Ekong et al., 2009).

Forced swim test. Rats were forced to swim in an ines-

capable situation, they tend to become immobile after

initial vigorous activity. This immobility has been

described as a symptom of a behavioural despair

(Porsolt et al., 1977, 1978). The test was conducted

using a modification of the method of Porsolt. Briefly,

rats were individually placed in a 80-cm high and

40 cm diameter plastic cylindrical tank containing

50 cm of water with ambient temperature (23�C), so

that the rat’s hindlimbs could not reach the tank’s

floor. Rats were allowed to swim for 6 min and their

activity was tracked (Karolewicz and Paul, 2001).

The videotaped behaviour was subsequently analyzed

Doguc et al. 3



by a rater who was blind to the animals’ housing con-

ditions. Duration of immobility was defined as when

the rat was stationary and only made the minimal

movements necessary to stay afloat, and mobility was

defined as swimming, jumping, rearing, sniffing or

diving which were considered active, escape-

directed behaviour. All of these movements were

tracked using an overhead camera (Sony SSC-

DC398P) interfaced with a computer which were

using the Smart Version 2.0 program.

Statistical analysis

The statistical analyses were carried out using the

SPSS for Windows program. The water maze data,

latency to escape and probe trial data, open-field data

and forced swim test data were first assessed by

Levene homogeneity test, and the data were not

homogeneous so the nonparametric tests were used

and results were expressed as the mean + SEM (stan-

dard error of mean) values. First, all the groups’ per-

formance were assessed by Friedmann test and

significance was found. A p value of less than 0.05

was considered to be statistically significant. To find

which data was responsible for the significance,

Wilcoxon signed ranks test was applied. Statistical

significance between groups was analyzed by

Krukal–Wallis test. The group comparisons of latency

to escape to the hidden platform data and probe data

of water maze and visible platform latency data were

assessed by Kruskal–Wallis test. Open-field test and

forced swim test data were again assessed by

Kruskal–Wallis test. We used Bonferroni corrected

Mann–Whitney U tests to determine which group was

responsible for the difference. A p value of less than

0.01 was considered to be statistically significant.

After the quartette comparisons, an additional statisti-

cal analysis was done, FEG and MEG were together

accepted as experiment group, FCG and MCG

were together accepted as control group and MWM

data, open-field data and FST data of these two

groups were assessed by one-way analysis of

variance. A p value of less than <0.05 was consid-

ered to be statistically significant.

Results

Morris water maze data

The groups’ performance was assessed by Friedmann

test, and it was found to be significant (p < 0.05).

Comparison of day performances was as follows: per-

formance of days 1–2, days 1–3, days 1– 4 and days

1–5 showed statistically significant differences in all

groups (p < 0.01; Figure 1). These results showed us

that all groups learnt the system and developed to

an acquisition level. There were no statistically signif-

icant difference between groups for the data of

latency to escape to the hidden platform per day and

there was no statistically significant difference

between groups for probe trial test (p > 0.05; Figure

2). Latency to escape to the visible platform was not

statistically significant (p > 0.05; Figure 3). So the

data that evaluated the hippocampus-dependent

0

10

20

30

40

50

60

70

80

90

Day 1 Day 2 Day 3 Day 4 Day 5

Tim

e (s

)

FEG

FCG

MEG

MCG

Figure 1. Day-to-day performances of the groups to findthe hidden platform. Acquisition period of hidden versionof the Morris water maze for the groups. Mean of escapelatency values per day was given. A p value of less than0.05 was considered to be statistically significant. Perfor-mance of days 1– 2, days 1–3, days 1–4 and days 1– 5showed statistically significant difference (p < 0.01).

0

5

10

15

20

25

30

35

40

FEG FCG MEG MCG

Tim

e (s

)

Figure 2. Probe trial performance of groups. During theprobe trial on day 6 when the platform had been removed,the time spent in the target quadrant where the platformhad been during acquisition period. A p value of less than0.05 was considered to be statistically significant.




spatial learning and memory showed no significant

difference between groups. When we compare the

MWM data as experiment and control group, like the

quartette comparisons performance of days 1–2, days

1–3 and days 1–4 showed statistically significant dif-

ference (p < 0.05). Day 1, day 2, day 3, day 4 and day

5 comparisons between control and experiment group

were not found to be statistically significant

(p > 0.05), so the spatial learning rates showed no dif-

ference between groups.

Open-field test data

Open-field maze data included number of line

crosses, rearing, centre crosses, edge duration and

centre duration. Kruskal–Wallis test was used to com-

pare these data of the groups. Line crosses and edge

duration data were statistically significant (p < 0.05).

Number of line crosses data of FEG was significantly

increased when compared with MEG and MCG

(p < 0.01) and also FCG’s line crosses data were signif-

icantly higher when compared with MCG (Table 1).

These data showed that the measure of locomotor

activity as well as exploration in female offspring was

significantly higher. When we compare control group

(FCG þ MCG, n ¼ 20) and experiment group

(FEG þMEG, n ¼ 20) to find whether the sex would

be the only fact of this significance, pairwise compar-

ison of the groups as control and experiment group

showed that the number of line crosses data still signif-

icant, and the number of line crosses was significantly

higher in experiment group when compared with con-

trol group (p < 0.05; Table 2). So AFCA exposition sig-

nificantly increased the exploration behaviour and

locomotor activity for this data. Edge duration time

was significantly decreased in MEG when compared

with MCG (p < 0.01; Table 1). According to this data,

we should mention that in male offspring the AFCA

seems to lower the anxiety; but in females, there was

no significant difference. But comparison of groups

as experiment (n ¼ 20) and control group (n ¼ 20),

unlike the comparison of four group edge duration data

was not significant (p > 0.05), but the number of wall-

ing data was significant (p < 0.05; Table 2). Number of

walling was significantly higher in experiment group

when compared with control group, these data support

the number of line crosses, as they are usually used as

measure of locomotor activity, as well as higher explo-

ration and lower anxiety for experiment group.

In quartette comparisons not only AFCA’s effect

but the contribution of sex to locomotor behaviour

was also evaluated. We can mention that gender

should be an important fact on the differences in loco-

motor activity, exploration, and anxiety behaviour.

But statistical analysis of data as experiment and con-

trol group showed us the significance in the number of

line crosses and number of walling data. So AFCA

alone effected two behaviours which we used as the

signs of explorative and higher locomotor behaviour.

In addition, AFCA and gender together might display

synergistic effect on locomotor behaviour and might

create different effects on male or female gender.

Forced swim test data

Comparisons of mobility (active behaviour) and

immobility period of the groups showed no statisti-

cally significant difference from each other

(p > 0.05; Figure 4). In addition, there were no signif-

icant difference (p > 0.05) when we compared the

experiment (n ¼ 20) and control groups (n ¼ 20).

So the rats were considered as active, and they

showed escape-directed behaviour.

Discussion

Our results showed that commonly used artificial food

colours have no adverse effects on hippocampus-

dependent spatial learning and memory when applied

as a mixture with their ADI amounts. In the assess-

ment of locomotor activity as well as exploration and

anxiety, artificial food colours mixture showed a few

significant effects. Quartette comparisons gave rise to

the thought that the gender has an important contribu-

tion on significance. Females seem to have signifi-

cantly better exploratory behaviour and also have

better locomotor activity. But comparison of experi-

ment and control groups showed that locomotor

0

2

4

6

8

10

12

14

16

18T

ime

(s)

FEG FCG MEG MCG

Figure 3. Visible platform performance of the groups. Visi-ble platform version of Morris water maze. A p value of lessthan 0.05 was considered to be statistically significant.

Doguc et al. 5



activity and exploration behaviour were higher in

experiment group so not gender alone but artificial

food colour mixture and gender together may have

synergistic effect on locomotor activity and explora-

tion behaviour.

Tanaka studied different AFCAs and evaluated

selected reproductive and neurobehavioural para-

meters in mice. Different doses of erythrosine, sunset

yellow FCF, Ponceau 4R, amaranth, allura red AC

and lac dye in the diet were administered. In the study

with erythrosine, emotional and movement activities

were significantly increased in a dose-related manner

and also in T-maze, movement time and average

speed were significantly increased in the high-dose

group in females (Tanaka, 2001). For sunset yellow

FCF, some of the different neurobehavioural para-

meters were affected in female and in male offspring

during the early lactation period in a dose-related

manner. The middle- and high-dose levels of sunset

yellow FCF produced some adverse effects in repro-

ductive and neurobehavioural parameters (Tanaka,

1996). For Ponceau 4R, in multiple water T-maze per-

formance in the F1 generation, the time taken was sig-

nificantly longer than the control in the middle-dose

and high-dose groups in males, and those effects were

significantly dose related (Tanaka, 2006). For middle

doses of amaranth, several parameters of movement

activity of male offspring at 3 weeks of age were

affected (Tanaka, 1993). There were few adverse

effects of allura red AC on either movement activity

or maze learning in F1 generation mice in each sex

(Tanaka, 1994). The natural colour additive, lac dye,

had also affected the neurobehavioural parameters.

Swimming head angle was significantly affected

in male offspring and olfactory orientation was

significantly accelerated in female offspring in a

dose-related manner (Tanaka, 1997). In Tanaka’s

Table 2. Open-field test data comparison as experiment and control groupa,b

GroupsNumber of line

crossesNumber of

wallingNumber of

rearingNumber of

centre crossesEdge

duration (s)Centre

duration (s)

Experiment group(FEG þ MEG; n ¼ 20)

117.54 + 26.11c 9.78 + 4.55c 2.83 + 2.49 1.46 + 1.21 231.67 + 63.85 4.29 + 3.97

Control group (FCG þMCG; n ¼ 20)

96.83 + 33.66d 7.5 + 3.14d 2.52 + 2.04 1.26 + 1.18 263.00 + 53.10 3.17 + 2.79

aData are given as mean + SD. A p value of less than 0.05 was considered to be statistically significant. Data marked with ‘c’ arestatistically significant when compared with ‘d’ (p < 0.05).bNumber of line crosses and number of walling data are significantly increased in experiment group when compared with the controlgroup (p < 0.05).

Table 1. Open-field test data of the groupsa,b

GroupsNumber of line

crossesNumber of

wallingNumber of

rearingNumber of centre

crossesEdge

duration (s)Centre

duration (s)

FEG (n ¼ 10) 131.42 + 6.65c 10.91 + 1.34 4.17 + 1.24 1.17 + 0.39 244.08 + 21.55 2.58 + 0.93FCG (n ¼ 10) 118.73 + 6.87x 8.25 + 0.85 3.45 + 0.87 1.82 + 0.42 244.45 + 13.99 3.82 + 1.14MEG (n ¼ 10) 103.67 + 6.28d 8.75 + 1.31 1.5 + 0.53 1.75 + 0.31 219.25 + 14.77c 3.50 + 1.16MCG (n ¼ 10) 76.75 + 8.53d,y 6.75 + 0.95 1.67 + 0.48 0.75 + 0.35 281.58 + 15.71d 2.58 + 1.19

aData are given as mean + SEM. A p value of less than 0.01 was considered to be statistically significant. Data marked with ‘c’ arestatistically significant when compared with ‘d’ and ‘x’ is statistically significant when compared with ‘y’ (p < 0.01).bNumber of line crosses data is significantly higher in FEG when compared with MEG and MCG. In addition, FCG data are significantlyhigher when compared with the MCG. Edge duration data decreased significantly in MEG when compared with MCG (p < 0.01).

0

50

100

150

200

250

Tim

e (s

) ImmobilityTime

MobilityTime

FEG FCG MEG MCG

Figure 4. Forced swim test performances of the groups.Mobility and immobility periods of the groups were plotted.A p value of less than 0.05 was considered to be statisticallysignificant.




studies, AFCA’s adverse effects on neurobehavioural

parameters seemed to be dose related, and adverse

effects at selected parameters were seen with the middle

and high doses which were greater than human ADI.

Also different doses caused different effects in male and

female mice. In accordance with our results; in Tanaka’s

studies gender was reported as an important fact with the

AFCAs’ effect on neurobehavioural parameters.

On the other hand, Sobotka et al. (1977) studied the

different doses of artificial food colour tartrazine dur-

ing gestation and lactation and showed no significant

effect on the developing nervous system.

Studies in children are still inconsistent. Several

studies reported improved behavioural characteristics

in part, but not in all children of their study population

(Conners et al., 1976; Kaplan et al., 1989; Williams et

al., 1978), whereas others did not provide support for

the Feingold hypothesis (Harley et al., 1978a, b). Sil-

ver (1986) claimed that the specific elimination of

synthetic food colours from the diet did not appear

to be a major factor in the reduction of hyperactive

behaviour in the majority of children. A recent study

by McCann et al. (2007) has concluded that exposure

to two mixtures of four synthetic colours plus a

sodium benzoate preservative in the diet resulted in

increased hyperactivity in 3-year-old and 8- to 9-

year-old children in general population and presented

a support for the case that food additives exacerbate

hyperactive behaviours (inattention, impulsivity and

overactivity) in children at least up to middle child-

hood. These findings also showed that adverse effects

are not just seen in children with extreme hyperactiv-

ity (i.e. ADHD) but can also be seen in the general

population and across the range of severities of

hyperactivity (McCann et al., 2007). The EFSA panel

concludes that the study of Mc Cann et al. provided

limited evidence that the two different mixtures of syn-

thetic colours and sodium benzoate tested had a small

and statistically significant effect on activity and atten-

tion in some children selected from the general popu-

lation, although the effects were not observed for all

children in all age groups and were not consistent for

the two mixtures. The findings may thus be relevant for

specific individuals within the population, showing

sensitivity to food additives in general or to food col-

ours in particular (The EFSA Panel, 2008).

The results of research carried on human and

animals do not overlap completely. The selected tasks

that were used to evaluate the neurobehavioural

effects in experimental studies were not directly

associated with ADHD, but they provide the signs

of increased locomotor activity and spatial learning

difficulty. Although there were some significant

effects on locomotor activity as well as exploration

and anxiety, artificial food colours which were used

with ADI values during pregnancy had no adverse

effect on spatial working memory and did not create

a depressive behaviour in offspring. In our study ADI

values of artificial food colours have been used as a

mixture, so we cannot ignore the possibility that

exceeding ADI values might cause additional and

increased adverse effects. Thus, we can suggest that

although gender seems to be an additional fact in the

side effects of artificial food colours, we are not able

to conclude exact clinical results based on these data.

Besides in today’s world too much of artificial foods

are being consumed and the amounts that exceed ADI

values may become a question. Therefore, it would be

a reliable approach to lower the consumption of

take-home foods that contain AFCAs.

Authors’ note

This manuscript has been presented as a poster at the XI

National Congress of Clinical Biochemistry, which was

held in Antalya between 28th April and 1st May 2011.

Conflict of interest

The authors declared no conflicts of interest.

Funding

This research was supported by Scientific Research Fund of

Suleyman Demirel University.

References

Aguiar A, Eubig PA and Schantz SL (2010) Review: atten-

tion deficit/hyperactivity disorder: a focused overview

for children’s environmental health researchers. Envi-

ronmental Health Perspectives 118(12): 1646–1653.

Bellisle F (2004) Review: effects of diet on behaviour and

cognition in children. British Journal of Nutrition

92(suppl 2): S227–S332.

Brotto LA, Alasdair MB and Boris BG (2000) Sex differ-

ences in forced-swim and open-field test behaviours

after chronic administration of melatonin. European

Journal of Pharmacology 402: 87–93.

Conners CK, Goyette CH, Southwick DA, Lees JM and

Andrulonis PA (1976) Food additives and hyperkinesis:

a controlled double blind experiment. Pediatrics 58:

154–166.

EFSA Panel (2008) Assessment of the results of the study

by McCann et al. (2007) on the effect of some colours

and sodium benzoate on children’s behaviour. The

EFSA Journal 660: 1–54.

Doguc et al. 7



Ekong MB, Mesembe OE, Bisong SA and Ekeoma AO

(2009) Neurobehavioural activity in albino Wistar rats

in the open field maze following long term tobacco diet

ingestion. The Internet Journal of Neurology 10(2).

Feingold BF (1976) Hyperkinesis and learning disabilities

linked to the ingestion of artificial food colors and

flavors. Journal of Learning Disabilities 9: 19–27.

Harley JP, Mattews CG and Eichman P (1978a) Synthetic

food colours and hyperactivity in children: a double-

blind challenge experiment. Pediatrics 61: 975–983.

Harley JP, Ray RS, Tomasi L, Eichman PL, Matthews CG,

Chun R, et al. (1978b) Hyperkinesis and food additives:

testing the Feingold hypothesis. Pediatrics 61: 818–828.

Kaplan BJ, McNicol J, Conte RA and Moghadam HK

(1989) Dietary replacement in preschool-aged hyperac-

tive boys. Pediatrics 83: 7–17.

Karolewicz B, Paul IA (2001) Short communication; group

housing of mice increases immobility and antidepressant

sensitivity in the forced swim and tail suspension tests.

European Journal of Pharmacology 415: 197–201.

McCann D, Barrett A, Cooper A, Crumpler D, Dalen L,

Grimshaw K, et al. (2007) Food additives and hyper-

active behaviour in 3-year-old and 8/9-year-old

children in the community: a randomised, double-

blinded, placebo-controlled trial. Lancet. 370(9598):

1560–1567.

Morris R (1984) Developments of a water-maze procedure

for studying spatial learning in the rat. Journal of

Neuroscience Methods 11: 47–60.

Porsolt RD, Anton G, Blaret N and Jalfre M (1978)

Behavioural despair in rats: a new model sensitive to

antidepressant treatments. European Journal of

Pharmacology 47: 379–391.

Porsolt RD, Bertin A and Jalfre M (1977) Behavioral

despair in mice: a primary screening test for antidepres-

sants. Archives Internationales de Pharmacodnamie et

de Therapie 229: 327–336.

Silver LB (1986) The ‘magic cure’: a review of the current

controversial approaches for threatening learning disabil-

ities. American Journal of Diseases 140: 1045–1052.

Sobotka TJ, Brodie RE and Spaid SL (1977) Tartrazine and

the developing nervous system of rats. Journal of

Toxicology and Environmental Health 2(5): 1211–1220.

Tanaka T (1993) Reproductive and neurobehavioral effects

of amaranth administered to mice in drinking water.

Toxicology & Industrial Health 9(6): 1027–1035.


of Allura Red AC administered to mice in the diet. Tox-

icology 92(1-3): 169–177.


of Sunset yellow FCF administered to mice in the diet.

Toxicology & Industrial Health 12(1): 69–79.

Tanaka T (1997) Reproductive and neurobehavioural

effects of lac dye administered in the diet to mice. Food

Additives & Contaminants 14(4): 373–380.

Tanaka T (2001) Reproductive and neurobehavioural

toxicity study of erythrosine administered to mice

in the diet. Food and Chemical Toxicology 39(5):

447–454.

Tanaka T (2006) Reproductive and neurobehavioural toxicity

study of Ponceau 4R administered to mice in the diet. Food

and Chemical Toxicology 44(10): 1651–1658.

Williams JI, Cram DM, Tausig FT and Webster E

(1978) Relative effects of drugs and diet on hyperac-

tive behaviours: an experimental study. Pediatrics 61:

811–817.




Documents

Effects of maternally exposed colouring food additives on cognitive performance in rats