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Reproductive and neurobehavioural toxicity study of tartrazine administered to mice in the diet
Food and Chemical Toxicology 44 (2006) 179–187 www.elsevier.com/locate/foodchemtox
Reproductive and neurobehavioural toxicity study of tartrazine administered to mice in the diet Toyohito Tanaka
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Department of Environmental Health and Toxicology, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan Received 12 January 2005; accepted 27 June 2005
Abstract Tartrazine was given in the diet to provide levels of 0% (control), 0.05%, 0.15%, and 0.45% (approximately 83, 259, 773 mg/kg/ day, respectively) from five weeks of age of the F0 generation to nine weeks of age of the F1 generation in mice, and selected reproductive and neurobehavioural parameters were measured. In movement activity of exploratory behaviour in the F0 generation, number of vertical activity was significantly increased in the middle-dose group in males. There were no adverse effects of tartrazine on either litter size, litter weight and sex ratio at birth. The average body weight of male offspring was significantly increased in the highdose group and that of female offspring was significantly increased in the middle-dose group at birth. In behavioural developmental parameters, surface righting at PND 4 was significantly accelerated in the high-dose group in male offspring, and those effects were significantly dose-related in a trend test (P < 0.01). Cliff avoidance at PND 7 was significantly accelerated in the middle-dose group in male offspring. Negative geotaxis at PND 4 was significantly delayed in the high-dose group in female offspring. Other variables measured showed no significant adverse effects in either sex in the lactation period. In movement activity of exploratory behaviour in the F1 generation, number of movement showed a significant tendency to be affected in the treatment groups in male offspring in a trend test (P < 0.05). The dose level of tartrazine in the present study produced a few adverse effects in neurobehavioural parameters during the lactation period in mice. Nevertheless, the high-dose level were in excess of the ADI of tartrazine (0–7.5 mg/kg bw), and the actual dietary intake of tartrazine is presumed to be much lower. It would therefore appear that the levels of actual dietary intake of tartrazine is unlikely to produce any adverse effects in humans. 2005 Elsevier Ltd. All rights reserved. Keywords: Behavioural development; Tartrazine; Food dye; Maze learning; Mice; Movement activity; Reproductive toxicity
1. Introduction The color additives, tartrazine, FD&C Yellow No. 5, C.I. No. 19140, is principally the trisodium 5-hydroxy-1(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-H-pyrazol3-carboxylate. Tartrazine is an orange-colored, water soluble powder widely used in food products, drugs, cosmetics and pharmaceuticals. The estimated amounts of tartrazine manufactured in 1996 were approximately
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0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.06.011
71.35 t in Japan and 985.76 t in USA (Ishimitsu et al., 1998). The acceptable daily intake (ADI) of human is 0–7.5 mg/kg bw (JECFA, 1996). In toxicological studies of tartrazine, Davis et al. (1964) reported that the incidence of tumors and of the common incidental diseases was unaffected by tartrazine in the diet (0.5–5.0%) in chronic toxicity study (two years) of rats. Maekawa et al. (1987) found that tartrazine in drinking water (1.0–2.0%) showed no carcinogenic effects in two years toxicity study of rats. Borzelleca and Hallagan (1988a) reported that the no observed adverse effect levels (NOAEL) of tartrazine were 5.0% (2641 and 3348 mg/kg/day for males and
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females in rats, respectively) in chronic toxicity study (two years) via utero. Borzelleca and Hallagan (1988b) reported that the NOAEL of tartrazine in mice were 5.0% (8103 and 9735 mg/kg/day for males and females, respectively) in chronic toxicity study (104 weeks). As regards reproductive and developmental toxicity studies, Collins et al. (1990) found that tartrazine produced neither toxic nor teratogenic effects in rats by gavage (60–1000 mg/kg/day). Also, Collins et al. (1992) reported that tartrazine produced no teratogenic effects in drinking water (0.05–0.7%) in rats. As regards behavioural toxicity studies, Sobotka et al. (1977) reported that tartrazine exerted minimal neurobehavioural effects (only neuromotor development in females) in rats (1.0–2.0% in the diet). Also, as regards hyperactivity of children, Rowe and Rowe (1994) reported that behavioural changes in irritability, restlessness, and sleep disturbance were associated with the ingestion of tartrazine in some children, and those effects showed dose-related (1–50 mg/child). Ward (1997) reported that 23 children consumed a tartrazine beverage showed increased levels of overactivity, aggressive and/or violent activity, poor speech, poor coordination, and the development of asthma and/or eczema. Nevertheless, there were no studies on reproductive and neurobehavioural toxicity of tartrazine in animal species. Therefore, the present study was designed to evaluate reproductive and neurobehavioural effects of tartrazine in mice throughout two-generations.
group (20 mice: 10/sex) were given the basal diets (Nihon Clea, CE-2) for the corresponding time period. Individual food intake of mice was measured during five divided periods: preconception (from five weeks of age to mating), mating (five days), gestation (14 days), lactation (from birth to weaning), and F1 generation (4–9 weeks of age).
2. Materials and methods
2.5. Neurobehavioural procedure
2.1. Materials
The functional and behavioural developmental parameters were measured and scored for all individual offspring during the lactation period in the F1 generation (Tanaka et al., 1992), and were analyzed on score frequencies (Tanaka, 1995). The measured variables were as follows:
Tartrazine (Food Yellow No. 4) was obtained from Tokyo Kasei Co., Ltd., Tokyo, Japan (Lot No. GL 01). The purity of the chemical was more than 85.0%.
2.4. Reproductive procedure The animals from the F0 generation were five weeks of age at the start of the study. The animals were individually weighed at experimental day 0, 2, 4, 7, 14, 21, 28, and 30 during the preconception period. At nine weeks of age, each female was paired with one male from the same treatment group, for a period of five days. The males were removed from females after five days, and the females were allowed to carry their litters to term, deliver, and rear all of their offspring. In the F1 generation, litter size, litter weight, and sex ratio (male/female) were measured on postnatal day (PND) 0 (at birth). The offspring were individually weighed on PNDs 0, 4, 7, 14, and 21 during the lactation period. The survival indices were calculated as (live offspring at each period)/(live and dead offspring at birth) · 100(%). The offspring were weaned when they were four weeks of age, and one male and one female were randomly selected to continue treatment from each litter. The animals were individually weighed at 4–9 weeks of age after weaning.
2.2. Animals and maintenance Male and female mice (Crj: CD-1, four weeks of age) were purchased from Charles River Japan Inc., Kanagawa, Japan. They were individually housed in polycarbonate solid-floored cages with wood flakes, and kept in a temperature controlled room maintained at 25 ± 1 C with relative humidity of 50 ± 5% on a 12 h light/dark cycle. They were given control or experimental diets and water ad libitum. 2.3. Experimental design Tartrazine was administered in the diet to 60 mice (10/sex/group) at dietary levels of 0.05%, 0.15%, and 0.45%, from five weeks of age of the F0 generation to nine weeks of age of the F1 generation. The control
1. Surface righting on PNDs 4 and 7 (Fox, 1965; Pantaleoni et al., 1988). The offspring were placed on their backs on a smooth surface and the time required to right themselves to a position where all four limbs touched the surface was recorded. The scoring rate for successful righting was: 2 = righting within 1 s; 1 = more than 1 s but within 2 s; 0 = more than 2 s. 2. Negative geotaxis on PNDs 4 and 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988). The offspring were placed in a head-down position on a 30 inclined plane and the time required to reorient to a head-up position was recorded. The plane was made of plywood covered with sandpaper (fine grade). The following scoring rate was employed: 0 = no response within 60 s; 1 = response within 60 s; 2 = response within 30 s.
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3. Cliff avoidance on PND 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988). The offspring were placed on a platform elevated 10 cm above a table top. The forelimbs and snout of the animals were positioned so that the edge of the platform passed just behind an imaginary line drawn between the eye orbits. The following scoring rate was employed: 0 = no response within 20 s; 1 = avoided backwards within 20 s, 2 = avoiding with turn. 4. Swimming behaviour on PNDs 4 and 14 (Fox, 1965; Pantaleoni et al., 1988). The offspring were placed into a tank with water temperature maintained at 23 ± 1 C and swimming behaviour was rated for direction (straight = 3, circling = 2, floating = 1) and head angle (ears out of water = 4, ears half out of water = 3, nose and top of head out of water = 2, and unable to hold head-up = 1). Limb movement was rated as either 1 = all four limbs used, or 2 = hindlimbs only used. 5. Olfactory orientation on PND 14 (Altman and Sudarshan, 1975; Barlow et al., 1978; Meyer and Hansen, 1980). The offspring were placed in the arm of an apparatus consisting of two compartment connected by the arm. One compartment was covered with home wood flakes from their cages and the other was covered with fresh wood flakes. The time required to enter the compartment with home wood flakes was recorded. The following scoring rate was employed: 0 = no response within 90 s; 1 = entered the home wood flakes compartment via the fresh wood flakes compartment; 2 = entered the home wood flakes compartment directly. Exploratory behaviour of mice was measured in an animal movement analyzing system ANIMATE AT420 (Toyo Sangyo Co., Ltd., Toyama, Japan) at eight weeks of age in the F0 generation and at three and eight weeks of age in the F1 generation. The system consisted of a doughnut-shaped cage made from acrylate resins with 36 units of detectors of near-infrared photosensors for measuring spontaneous motor activity (Matsumoto et al., 1990a,b). The behavioural variables were recorded for 10 min on all animals at eight weeks of age in the F0 and F1 generations and on one male and one female selected randomly from each litter at three weeks of age in the F1 generation. The variables measured were: number of movements, movement time (s), number of horizontal activities, total distance (cm), number of vertical activities, vertical time (s), number of turnings, average distance (cm), average speed (cm/s), and number of defecation. The animals each performed one trial a day for three days in a multiple-T water maze of BielÕs type adapted for mice at seven weeks of age in the F1 generation (Biel, 1940; Kitatani et al., 1988). The water temperature was maintained at 20 ± 1 C. The time taken and number of
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errors were measured from the start to finish for a maximum of 120 s. If the time taken was greater than 120 s, it was recorded as 120 s (Kitatani et al., 1988). 2.6. Statistical analysis Food intake, litter size, litter weight, and body weight were assessed with the BonferroniÕs multiple comparison test after the analysis of variance (ANOVA) or the Kruskal–Wallis test. Sex ratio, survival and behavioural developmental data were assessed with the v2 test or the FisherÕs exact test of frequency analysis. Movement activity data were assessed with the Steel–Dwass test of non-parametric methods (Martin and Bateson, 1990). Multiple-T water maze performance data were assessed with the Sign–Wilcoxon test for trials and assessed with the Steel–Dwass test within each treatment group. Dose–response effects were assessed with the Jonckheere test for ordered alternatives or the cumulative v2 test (multi) for frequency data.
3. Results 3.1. Food and chemical intake There were no significant effects of tartrazine on the average food intake during any periods (Table 1). Therefore, the chemical intake was consistently increased in a dose-related manner during each period (Table 1). 3.2. F0 generation The average body weight of male and female mice showed no significant adverse effects during the preconception or mating periods, and the average body weight of dams showed no significant adverse effects during the gestation and lactation periods. In movement activity of exploratory behaviour, number of vertical activity in males was significantly increased in the middle-dose group (Fig. 1). Other variables of measurement showed no significant adverse effects of tartrazine in either sex. Each one female had no became pregnant in the control and high-dose groups, and abortion was observed in six dams, one dam each in the control and middle-dose groups, and two dams each in the low- and high-dose groups (Table 2). During the first week of the lactation period, one dam of the high-dose group showed underdeveloped mammary glands, which were observed as underdeveloped mammalae. One dam of the middledose group died during the second week of the lactation period, and one dam of the control group died during the third week of the lactation period.
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Table 1 Average daily food and chemical intake of mice administered tartrazine in two-generation toxicity study Dose level (%)
0
0.05
0.15
0.45
Food intake (g/kg/day) F0 generation Preconception Male Female Mating Gestation Lactation
153.9 ± 11.00 182.0 ± 16.43 141.9 ± 22.69 157.6 ± 20.96 558.1 ± 57.61
146.2 ± 6.98 192.7 ± 23.08 136.3 ± 15.39 169.5 ± 22.81 576.8 ± 55.35
159.4 ± 10.58 197.0 ± 39.88 149.0 ± 24.34 179.6 ± 48.97 551.7 ± 107.66
159.8 ± 12.07 191.0 ± 22.04 143.4 ± 16.86 162.0 ± 16.14 564.6 ± 91.05
F1 generation Male Female
160.8 ± 9.20 180.3 ± 8.75
166.6 ± 11.71 188.4 ± 20.47
173.0 ± 15.78 176.8 ± 14.06
171.4 ± 13.66 202.7 ± 17.76
Chemical intake (mg/kg/day) F0 generation Preconception Male Female Mating Gestation Lactation
– – – – –
73.1 ± 3.49 96.4 ± 11.54 68.2 ± 7.69 84.8 ± 11.40 288.4 ± 27.67
239.2 ± 15.87 295.4 ± 59.82 223.5 ± 36.52 269.5 ± 73.45 827.6 ± 161.49
719.3 ± 54.32 859.5 ± 99.20 645.2 ± 75.89 728.9 ± 72.63 2540.8 ± 409.74
F1 generation Male Female
– –
83.3 ± 5.85 94.2 ± 10.24
259.6 ± 23.67 265.2 ± 21.09
771.3 ± 61.47 912.2 ± 79.94
Each value represents daily intake during each period (mean ± SD).
Fig. 1. Movement activity of exploratory behaviour at eight weeks of age of F0 generation male mice administered tartrazine in the diet. Each value represents the mean ± SE. Significantly different from controls: *P < 0.05.
Table 2 Summary of data of litters at birth in two-generation toxicity study of tartrazine administered to mice Dose level (%)
0
0.05
0.15
0.45
No. of females examined No. of pregnant females No. of litters No. of offspring Average litter size Average litter weight (g) Total sex ratio (male/female) Average sex ratio (male%)
10 9 8a 107a 13.4 ± 1.69a 20.78 ± 2.014 0.91 (51/56)a 47.9 ± 7.88a
10 10 8 105 13.1 ± 2.80 20.68 ± 3.733 0.78 (46/59) 44.3 ± 13.59
10 10 9 116 12.6 ± 2.80 20.40 ± 3.606 1.11 (61/55) 51.6 ± 15.59
10 9 7 90 12.9 ± 1.57 20.23 ± 2.592 0.96 (44/46) 49.4 ± 21.11
Each value represents the mean ± SD. a Including a litter killed by its dam at birth.
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3.3. F1 generation No significant adverse effects were observed in litter size, litter weight, or sex ratio at birth (Table 2). The average body weight of male offspring was significantly increased in the high-dose group and that of female offspring was significantly increased in the middle-dose group at birth (Table 3). Also, the average body weight of male offspring was significantly increased in the highdose group at PND 21 during the lactation period. One litter of the control group was killed by its dam at birth. One litter of the middle-dosed group was killed by its dam during the first week of the lactation period. One litter of the middle-dosed group died of accident of water supply during the first week of the lactation period. One litter of the high-dose group were decreased by undernourishment caused by underdeveloped mammary glands of dam during the lactation period. One litter of
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the middle-dose group was killed (to prevent them dying of starvation) in view of death of the dam during the second weeks of the lactation period. The survival indices showed a few significant adverse effect in the middledose group during the lactation period (Table 4). Regarding the behavioural developmental parameters, surface righting at PND 4 was significantly accelerated in the high-dose group in male offspring (Fig. 2), and those effects were significantly dose-related in a trend test (P < 0.01). Cliff avoidance at PND 7 was significantly accelerated in the middle-dose group in male offspring. Negative geotaxis at PND 4 was significantly delayed in the high-dose group in female offspring. Other variables measured showed no significant adverse effects in either sex in the lactation period. In movement activity of exploratory behaviour at three weeks of age, number of movement showed a significant tendency to be affected in the treatment groups in male offspring in
Table 3 Summary of average body weight (g) of offspring during the lactation period in two-generation toxicity study of tartrazine administered to mice Dose levels (%)
0
0.05
0.15
0.45
Male offspring PND 0 PND 4 PND 7 PND 14 PND 21
1.52 ± 0.138 2.90 ± 0.397 4.41 ± 0.607 6.70 ± 1.005 11.95 ± 2.187
1.61 ± 0.157 3.05 ± 0.402 4.64 ± 0.635 6.97 ± 1.088 13.06 ± 2.156
1.60 ± 0.175 3.01 ± 0.382 4.58 ± 0.686 6.66 ± 1.272 12.77 ± 2.649
1.63 ± 0.191* 3.10 ± 0.575 4.69 ± 0.673 7.09 ± 1.089 13.23 ± 1.936*
Female offspring PND 0 PND 4 PND 7 PND 14 PND 21
1.48 ± 0.127 2.83 ± 0.404 4.29 ± 0.562 6.45 ± 1.030 11.48 ± 1.999
1.55 ± 0.149 2.94 ± 0.378 4.45 ± 0.634 6.59 ± 1.102 11.88 ± 2.414
1.57 ± 0.131** 2.99 ± 0.389 4.57 ± 0.769 6.77 ± 1.559 12.17 ± 2.971
1.52 ± 0.126 2.65 ± 0.696 4.07 ± 1.107 6.55 ± 1.187 11.61 ± 2.546
Each value represents the mean ± SD. Significantly different from controls: *P < 0.05,
**P
< 0.01.
Table 4 Summary of number of offspring and survival index (%) during the lactation period in two-generation toxicity study of tartrazine administered to mice Dose level (%)
0
Male offspring PND 0 PND 4 PND 7 PND 14 PND 21
46 46 46 46 46
Female offspring PND 0 PND 4 PND 7 PND 14 PND 21
51 51 51 51 51
0.05
0.15
(90.2)a (90.2) (90.2) (90.2) (90.2)
46 46 46 45 44
(100.0) (100.0) (100.0) (97.8) (95.7)
61 55 49 48 37
(100.0)* (90.2) (80.3) (78.7) (60.7)***
44 43 42 42 41
(100.0) (97.7) (95.5) (95.5) (93.2)
(91.1)a (91.1) (91.1) (91.1) (91.1)
59 59 59 59 59
(100.0)* (100.0)* (100.0)* (100.0)* (100.0)*
55 47 40 40 35
(100.0) (85.5) (72.7)* (72.7)* (63.6)*
46 44 40 39 39
(100.0) (95.7) (87.0) (84.8) (84.8)
Each value represents number of offspring: survival index (%) in parentheses. Significant different from controls: *P < 0.05, **P < 0.01, ***P < 0.001. a Calculated with including a litter killed by its dam at birth.
0.45
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a trend test (P < 0.05) (Fig. 3). Other variables measured showed no adverse effects in either sex. There were some significant effects of tartrazine on multiple-T water maze performance at seven weeks of age. In males, time taken was significantly reduced on the second trials as compared to the first trial in the control and high-dose groups, and on the third trial as compared to the first trial in the low-dose group (Fig. 4). Nevertheless, there was no significant adverse effect on maze learning among treatment groups compared to controls in males. In females, time taken was significantly reduced on the third trial in the high-dose group as compared to the first trial (Fig. 5). Also, number of errors was significantly reduced on the third trial in the highdose group as compared to the first trial in females. Nevertheless, there was no significant adverse effect on maze learning among treatment groups compared to controls in females. For movement activity of exploratory behaviour at eight weeks of age, any variable measured showed no adverse effects of tartrazine in either sex. The average body weight of male and female mice showed no significant adverse effects after weaning. 4. Discussion
Fig. 2. Score frequencies for behavioural development in the lactation period in two-generation toxicity study of tartrazine administered to mice. Significantly different from controls: *P < 0.05, ***P < 0.001. Surface righting at PND 4 in male offspring showed a significantly dose-related manner in a trend test (P < 0.01).
In the present study, tartrazine showed a few significant adverse effects on neurobehavioural parameters. The average body weight of offspring was significantly increased in the high-dosed group at PND 21 in male offspring. Nevertheless, the average body weights of male offspring in the high-dosed group were significantly increased at birth, and showed a tendency to be increased at PNDs 4, 7 and 14 (not significant). It therefore seems that the differences of male offspring weight between the control and high-dosed groups were caused not by tartrazine treatment but by the body size at birth. The survival indices showed a few significant adverse
Fig. 3. Movement activity of exploratory behaviour at three weeks of age of F1 generation male offspring administered tartrazine in the diet. Each value represents the mean ± SE. Movement activity of male offspring showed a significantly dose-related manner in a trend test (P < 0.05).
T. Tanaka / Food and Chemical Toxicology 44 (2006) 179–187
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Fig. 4. Summary of multiple-T water maze performance at seven weeks of age of F1 generation male mice in two-generation toxicity study of tartrazine. Each value represents the mean ± SE. Significantly different from the first trial: *P < 0.05.
effect in the middle-dose group during the lactation period. However, two litters of the middle-dose group died of accidents. It therefore seems that tartrazine produced no adverse effects on offspring survivals during the lactation period. In behavioural developmental parameters, tartrazine produced a few statistically significant effects in the measured variables as compared to control group in mice. Surface righting, indicative of the coordinated movement, was significantly accelerated in the high-dose group in male offspring in the early lactation period, and those effects were significantly dose-related (P < 0.01). Nevertheless, that of female offspring showed no significant effects during the lactation period. It therefore seems that tartrazine may have different effects on the behavioural development in sexes. In male offspring, number of movement of exploratory behaviour at three weeks of age in the F1 generation was significantly affected in a dose-related manner. Nevertheless, any variable measured showed no adverse
effects of tartrazine in movement activity of exploratory behaviour at eight weeks of age in the F1 generation in either sex. It therefore seems that tartrazine influenced exploratory behaviour in juvenile male mice only. In humans, Rowe and Rowe (1994) reported that some children who ingested tartrazine showed hyperactivity, and those effects showed dose-related (1–50 mg/child). Also, Ward (1997) reported that 23 children consumed a tartrazine beverage showed hyperactivity. Nevertheless, exploratory behaviour of mice was restrained by tartrazine treatment in juvenile males. It therefore seems that the dose levels of tartrazine in the present study may not induce hyperactivity in mice. From results, the high-dose level (0.45% in the diet) of tartrazine in the present study produced a few adverse effects on several behavioural parameters. Nevertheless, the high-dose level (645–2541 mg/kg/day) were in excess of the ADI of tartrazine (0–7.5 mg/kg bw). The actual dietary intake of tartrazine in Japan is presumed to be much lower, approximately 0.66 lg/kg/day in 1982
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Fig. 5. Summary of multiple-T water maze performance at seven weeks of age of F1 generation female mice in two-generation toxicity study of tartrazine. Each value represents the mean ± SE. Significantly different from the first trial: *P < 0.05.
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Reproductive and neurobehavioural toxicity study of tartrazine administered to mice in the diet Toyohito Tanaka
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Department of Environmental Health and Toxicology, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan Received 12 January 2005; accepted 27 June 2005
Abstract Tartrazine was given in the diet to provide levels of 0% (control), 0.05%, 0.15%, and 0.45% (approximately 83, 259, 773 mg/kg/ day, respectively) from five weeks of age of the F0 generation to nine weeks of age of the F1 generation in mice, and selected reproductive and neurobehavioural parameters were measured. In movement activity of exploratory behaviour in the F0 generation, number of vertical activity was significantly increased in the middle-dose group in males. There were no adverse effects of tartrazine on either litter size, litter weight and sex ratio at birth. The average body weight of male offspring was significantly increased in the highdose group and that of female offspring was significantly increased in the middle-dose group at birth. In behavioural developmental parameters, surface righting at PND 4 was significantly accelerated in the high-dose group in male offspring, and those effects were significantly dose-related in a trend test (P < 0.01). Cliff avoidance at PND 7 was significantly accelerated in the middle-dose group in male offspring. Negative geotaxis at PND 4 was significantly delayed in the high-dose group in female offspring. Other variables measured showed no significant adverse effects in either sex in the lactation period. In movement activity of exploratory behaviour in the F1 generation, number of movement showed a significant tendency to be affected in the treatment groups in male offspring in a trend test (P < 0.05). The dose level of tartrazine in the present study produced a few adverse effects in neurobehavioural parameters during the lactation period in mice. Nevertheless, the high-dose level were in excess of the ADI of tartrazine (0–7.5 mg/kg bw), and the actual dietary intake of tartrazine is presumed to be much lower. It would therefore appear that the levels of actual dietary intake of tartrazine is unlikely to produce any adverse effects in humans. 2005 Elsevier Ltd. All rights reserved. Keywords: Behavioural development; Tartrazine; Food dye; Maze learning; Mice; Movement activity; Reproductive toxicity
1. Introduction The color additives, tartrazine, FD&C Yellow No. 5, C.I. No. 19140, is principally the trisodium 5-hydroxy-1(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-H-pyrazol3-carboxylate. Tartrazine is an orange-colored, water soluble powder widely used in food products, drugs, cosmetics and pharmaceuticals. The estimated amounts of tartrazine manufactured in 1996 were approximately
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0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.06.011
71.35 t in Japan and 985.76 t in USA (Ishimitsu et al., 1998). The acceptable daily intake (ADI) of human is 0–7.5 mg/kg bw (JECFA, 1996). In toxicological studies of tartrazine, Davis et al. (1964) reported that the incidence of tumors and of the common incidental diseases was unaffected by tartrazine in the diet (0.5–5.0%) in chronic toxicity study (two years) of rats. Maekawa et al. (1987) found that tartrazine in drinking water (1.0–2.0%) showed no carcinogenic effects in two years toxicity study of rats. Borzelleca and Hallagan (1988a) reported that the no observed adverse effect levels (NOAEL) of tartrazine were 5.0% (2641 and 3348 mg/kg/day for males and
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females in rats, respectively) in chronic toxicity study (two years) via utero. Borzelleca and Hallagan (1988b) reported that the NOAEL of tartrazine in mice were 5.0% (8103 and 9735 mg/kg/day for males and females, respectively) in chronic toxicity study (104 weeks). As regards reproductive and developmental toxicity studies, Collins et al. (1990) found that tartrazine produced neither toxic nor teratogenic effects in rats by gavage (60–1000 mg/kg/day). Also, Collins et al. (1992) reported that tartrazine produced no teratogenic effects in drinking water (0.05–0.7%) in rats. As regards behavioural toxicity studies, Sobotka et al. (1977) reported that tartrazine exerted minimal neurobehavioural effects (only neuromotor development in females) in rats (1.0–2.0% in the diet). Also, as regards hyperactivity of children, Rowe and Rowe (1994) reported that behavioural changes in irritability, restlessness, and sleep disturbance were associated with the ingestion of tartrazine in some children, and those effects showed dose-related (1–50 mg/child). Ward (1997) reported that 23 children consumed a tartrazine beverage showed increased levels of overactivity, aggressive and/or violent activity, poor speech, poor coordination, and the development of asthma and/or eczema. Nevertheless, there were no studies on reproductive and neurobehavioural toxicity of tartrazine in animal species. Therefore, the present study was designed to evaluate reproductive and neurobehavioural effects of tartrazine in mice throughout two-generations.
group (20 mice: 10/sex) were given the basal diets (Nihon Clea, CE-2) for the corresponding time period. Individual food intake of mice was measured during five divided periods: preconception (from five weeks of age to mating), mating (five days), gestation (14 days), lactation (from birth to weaning), and F1 generation (4–9 weeks of age).
2. Materials and methods
2.5. Neurobehavioural procedure
2.1. Materials
The functional and behavioural developmental parameters were measured and scored for all individual offspring during the lactation period in the F1 generation (Tanaka et al., 1992), and were analyzed on score frequencies (Tanaka, 1995). The measured variables were as follows:
Tartrazine (Food Yellow No. 4) was obtained from Tokyo Kasei Co., Ltd., Tokyo, Japan (Lot No. GL 01). The purity of the chemical was more than 85.0%.
2.4. Reproductive procedure The animals from the F0 generation were five weeks of age at the start of the study. The animals were individually weighed at experimental day 0, 2, 4, 7, 14, 21, 28, and 30 during the preconception period. At nine weeks of age, each female was paired with one male from the same treatment group, for a period of five days. The males were removed from females after five days, and the females were allowed to carry their litters to term, deliver, and rear all of their offspring. In the F1 generation, litter size, litter weight, and sex ratio (male/female) were measured on postnatal day (PND) 0 (at birth). The offspring were individually weighed on PNDs 0, 4, 7, 14, and 21 during the lactation period. The survival indices were calculated as (live offspring at each period)/(live and dead offspring at birth) · 100(%). The offspring were weaned when they were four weeks of age, and one male and one female were randomly selected to continue treatment from each litter. The animals were individually weighed at 4–9 weeks of age after weaning.
2.2. Animals and maintenance Male and female mice (Crj: CD-1, four weeks of age) were purchased from Charles River Japan Inc., Kanagawa, Japan. They were individually housed in polycarbonate solid-floored cages with wood flakes, and kept in a temperature controlled room maintained at 25 ± 1 C with relative humidity of 50 ± 5% on a 12 h light/dark cycle. They were given control or experimental diets and water ad libitum. 2.3. Experimental design Tartrazine was administered in the diet to 60 mice (10/sex/group) at dietary levels of 0.05%, 0.15%, and 0.45%, from five weeks of age of the F0 generation to nine weeks of age of the F1 generation. The control
1. Surface righting on PNDs 4 and 7 (Fox, 1965; Pantaleoni et al., 1988). The offspring were placed on their backs on a smooth surface and the time required to right themselves to a position where all four limbs touched the surface was recorded. The scoring rate for successful righting was: 2 = righting within 1 s; 1 = more than 1 s but within 2 s; 0 = more than 2 s. 2. Negative geotaxis on PNDs 4 and 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988). The offspring were placed in a head-down position on a 30 inclined plane and the time required to reorient to a head-up position was recorded. The plane was made of plywood covered with sandpaper (fine grade). The following scoring rate was employed: 0 = no response within 60 s; 1 = response within 60 s; 2 = response within 30 s.
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3. Cliff avoidance on PND 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988). The offspring were placed on a platform elevated 10 cm above a table top. The forelimbs and snout of the animals were positioned so that the edge of the platform passed just behind an imaginary line drawn between the eye orbits. The following scoring rate was employed: 0 = no response within 20 s; 1 = avoided backwards within 20 s, 2 = avoiding with turn. 4. Swimming behaviour on PNDs 4 and 14 (Fox, 1965; Pantaleoni et al., 1988). The offspring were placed into a tank with water temperature maintained at 23 ± 1 C and swimming behaviour was rated for direction (straight = 3, circling = 2, floating = 1) and head angle (ears out of water = 4, ears half out of water = 3, nose and top of head out of water = 2, and unable to hold head-up = 1). Limb movement was rated as either 1 = all four limbs used, or 2 = hindlimbs only used. 5. Olfactory orientation on PND 14 (Altman and Sudarshan, 1975; Barlow et al., 1978; Meyer and Hansen, 1980). The offspring were placed in the arm of an apparatus consisting of two compartment connected by the arm. One compartment was covered with home wood flakes from their cages and the other was covered with fresh wood flakes. The time required to enter the compartment with home wood flakes was recorded. The following scoring rate was employed: 0 = no response within 90 s; 1 = entered the home wood flakes compartment via the fresh wood flakes compartment; 2 = entered the home wood flakes compartment directly. Exploratory behaviour of mice was measured in an animal movement analyzing system ANIMATE AT420 (Toyo Sangyo Co., Ltd., Toyama, Japan) at eight weeks of age in the F0 generation and at three and eight weeks of age in the F1 generation. The system consisted of a doughnut-shaped cage made from acrylate resins with 36 units of detectors of near-infrared photosensors for measuring spontaneous motor activity (Matsumoto et al., 1990a,b). The behavioural variables were recorded for 10 min on all animals at eight weeks of age in the F0 and F1 generations and on one male and one female selected randomly from each litter at three weeks of age in the F1 generation. The variables measured were: number of movements, movement time (s), number of horizontal activities, total distance (cm), number of vertical activities, vertical time (s), number of turnings, average distance (cm), average speed (cm/s), and number of defecation. The animals each performed one trial a day for three days in a multiple-T water maze of BielÕs type adapted for mice at seven weeks of age in the F1 generation (Biel, 1940; Kitatani et al., 1988). The water temperature was maintained at 20 ± 1 C. The time taken and number of
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errors were measured from the start to finish for a maximum of 120 s. If the time taken was greater than 120 s, it was recorded as 120 s (Kitatani et al., 1988). 2.6. Statistical analysis Food intake, litter size, litter weight, and body weight were assessed with the BonferroniÕs multiple comparison test after the analysis of variance (ANOVA) or the Kruskal–Wallis test. Sex ratio, survival and behavioural developmental data were assessed with the v2 test or the FisherÕs exact test of frequency analysis. Movement activity data were assessed with the Steel–Dwass test of non-parametric methods (Martin and Bateson, 1990). Multiple-T water maze performance data were assessed with the Sign–Wilcoxon test for trials and assessed with the Steel–Dwass test within each treatment group. Dose–response effects were assessed with the Jonckheere test for ordered alternatives or the cumulative v2 test (multi) for frequency data.
3. Results 3.1. Food and chemical intake There were no significant effects of tartrazine on the average food intake during any periods (Table 1). Therefore, the chemical intake was consistently increased in a dose-related manner during each period (Table 1). 3.2. F0 generation The average body weight of male and female mice showed no significant adverse effects during the preconception or mating periods, and the average body weight of dams showed no significant adverse effects during the gestation and lactation periods. In movement activity of exploratory behaviour, number of vertical activity in males was significantly increased in the middle-dose group (Fig. 1). Other variables of measurement showed no significant adverse effects of tartrazine in either sex. Each one female had no became pregnant in the control and high-dose groups, and abortion was observed in six dams, one dam each in the control and middle-dose groups, and two dams each in the low- and high-dose groups (Table 2). During the first week of the lactation period, one dam of the high-dose group showed underdeveloped mammary glands, which were observed as underdeveloped mammalae. One dam of the middledose group died during the second week of the lactation period, and one dam of the control group died during the third week of the lactation period.
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Table 1 Average daily food and chemical intake of mice administered tartrazine in two-generation toxicity study Dose level (%)
0
0.05
0.15
0.45
Food intake (g/kg/day) F0 generation Preconception Male Female Mating Gestation Lactation
153.9 ± 11.00 182.0 ± 16.43 141.9 ± 22.69 157.6 ± 20.96 558.1 ± 57.61
146.2 ± 6.98 192.7 ± 23.08 136.3 ± 15.39 169.5 ± 22.81 576.8 ± 55.35
159.4 ± 10.58 197.0 ± 39.88 149.0 ± 24.34 179.6 ± 48.97 551.7 ± 107.66
159.8 ± 12.07 191.0 ± 22.04 143.4 ± 16.86 162.0 ± 16.14 564.6 ± 91.05
F1 generation Male Female
160.8 ± 9.20 180.3 ± 8.75
166.6 ± 11.71 188.4 ± 20.47
173.0 ± 15.78 176.8 ± 14.06
171.4 ± 13.66 202.7 ± 17.76
Chemical intake (mg/kg/day) F0 generation Preconception Male Female Mating Gestation Lactation
– – – – –
73.1 ± 3.49 96.4 ± 11.54 68.2 ± 7.69 84.8 ± 11.40 288.4 ± 27.67
239.2 ± 15.87 295.4 ± 59.82 223.5 ± 36.52 269.5 ± 73.45 827.6 ± 161.49
719.3 ± 54.32 859.5 ± 99.20 645.2 ± 75.89 728.9 ± 72.63 2540.8 ± 409.74
F1 generation Male Female
– –
83.3 ± 5.85 94.2 ± 10.24
259.6 ± 23.67 265.2 ± 21.09
771.3 ± 61.47 912.2 ± 79.94
Each value represents daily intake during each period (mean ± SD).
Fig. 1. Movement activity of exploratory behaviour at eight weeks of age of F0 generation male mice administered tartrazine in the diet. Each value represents the mean ± SE. Significantly different from controls: *P < 0.05.
Table 2 Summary of data of litters at birth in two-generation toxicity study of tartrazine administered to mice Dose level (%)
0
0.05
0.15
0.45
No. of females examined No. of pregnant females No. of litters No. of offspring Average litter size Average litter weight (g) Total sex ratio (male/female) Average sex ratio (male%)
10 9 8a 107a 13.4 ± 1.69a 20.78 ± 2.014 0.91 (51/56)a 47.9 ± 7.88a
10 10 8 105 13.1 ± 2.80 20.68 ± 3.733 0.78 (46/59) 44.3 ± 13.59
10 10 9 116 12.6 ± 2.80 20.40 ± 3.606 1.11 (61/55) 51.6 ± 15.59
10 9 7 90 12.9 ± 1.57 20.23 ± 2.592 0.96 (44/46) 49.4 ± 21.11
Each value represents the mean ± SD. a Including a litter killed by its dam at birth.
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3.3. F1 generation No significant adverse effects were observed in litter size, litter weight, or sex ratio at birth (Table 2). The average body weight of male offspring was significantly increased in the high-dose group and that of female offspring was significantly increased in the middle-dose group at birth (Table 3). Also, the average body weight of male offspring was significantly increased in the highdose group at PND 21 during the lactation period. One litter of the control group was killed by its dam at birth. One litter of the middle-dosed group was killed by its dam during the first week of the lactation period. One litter of the middle-dosed group died of accident of water supply during the first week of the lactation period. One litter of the high-dose group were decreased by undernourishment caused by underdeveloped mammary glands of dam during the lactation period. One litter of
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the middle-dose group was killed (to prevent them dying of starvation) in view of death of the dam during the second weeks of the lactation period. The survival indices showed a few significant adverse effect in the middledose group during the lactation period (Table 4). Regarding the behavioural developmental parameters, surface righting at PND 4 was significantly accelerated in the high-dose group in male offspring (Fig. 2), and those effects were significantly dose-related in a trend test (P < 0.01). Cliff avoidance at PND 7 was significantly accelerated in the middle-dose group in male offspring. Negative geotaxis at PND 4 was significantly delayed in the high-dose group in female offspring. Other variables measured showed no significant adverse effects in either sex in the lactation period. In movement activity of exploratory behaviour at three weeks of age, number of movement showed a significant tendency to be affected in the treatment groups in male offspring in
Table 3 Summary of average body weight (g) of offspring during the lactation period in two-generation toxicity study of tartrazine administered to mice Dose levels (%)
0
0.05
0.15
0.45
Male offspring PND 0 PND 4 PND 7 PND 14 PND 21
1.52 ± 0.138 2.90 ± 0.397 4.41 ± 0.607 6.70 ± 1.005 11.95 ± 2.187
1.61 ± 0.157 3.05 ± 0.402 4.64 ± 0.635 6.97 ± 1.088 13.06 ± 2.156
1.60 ± 0.175 3.01 ± 0.382 4.58 ± 0.686 6.66 ± 1.272 12.77 ± 2.649
1.63 ± 0.191* 3.10 ± 0.575 4.69 ± 0.673 7.09 ± 1.089 13.23 ± 1.936*
Female offspring PND 0 PND 4 PND 7 PND 14 PND 21
1.48 ± 0.127 2.83 ± 0.404 4.29 ± 0.562 6.45 ± 1.030 11.48 ± 1.999
1.55 ± 0.149 2.94 ± 0.378 4.45 ± 0.634 6.59 ± 1.102 11.88 ± 2.414
1.57 ± 0.131** 2.99 ± 0.389 4.57 ± 0.769 6.77 ± 1.559 12.17 ± 2.971
1.52 ± 0.126 2.65 ± 0.696 4.07 ± 1.107 6.55 ± 1.187 11.61 ± 2.546
Each value represents the mean ± SD. Significantly different from controls: *P < 0.05,
**P
< 0.01.
Table 4 Summary of number of offspring and survival index (%) during the lactation period in two-generation toxicity study of tartrazine administered to mice Dose level (%)
0
Male offspring PND 0 PND 4 PND 7 PND 14 PND 21
46 46 46 46 46
Female offspring PND 0 PND 4 PND 7 PND 14 PND 21
51 51 51 51 51
0.05
0.15
(90.2)a (90.2) (90.2) (90.2) (90.2)
46 46 46 45 44
(100.0) (100.0) (100.0) (97.8) (95.7)
61 55 49 48 37
(100.0)* (90.2) (80.3) (78.7) (60.7)***
44 43 42 42 41
(100.0) (97.7) (95.5) (95.5) (93.2)
(91.1)a (91.1) (91.1) (91.1) (91.1)
59 59 59 59 59
(100.0)* (100.0)* (100.0)* (100.0)* (100.0)*
55 47 40 40 35
(100.0) (85.5) (72.7)* (72.7)* (63.6)*
46 44 40 39 39
(100.0) (95.7) (87.0) (84.8) (84.8)
Each value represents number of offspring: survival index (%) in parentheses. Significant different from controls: *P < 0.05, **P < 0.01, ***P < 0.001. a Calculated with including a litter killed by its dam at birth.
0.45
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a trend test (P < 0.05) (Fig. 3). Other variables measured showed no adverse effects in either sex. There were some significant effects of tartrazine on multiple-T water maze performance at seven weeks of age. In males, time taken was significantly reduced on the second trials as compared to the first trial in the control and high-dose groups, and on the third trial as compared to the first trial in the low-dose group (Fig. 4). Nevertheless, there was no significant adverse effect on maze learning among treatment groups compared to controls in males. In females, time taken was significantly reduced on the third trial in the high-dose group as compared to the first trial (Fig. 5). Also, number of errors was significantly reduced on the third trial in the highdose group as compared to the first trial in females. Nevertheless, there was no significant adverse effect on maze learning among treatment groups compared to controls in females. For movement activity of exploratory behaviour at eight weeks of age, any variable measured showed no adverse effects of tartrazine in either sex. The average body weight of male and female mice showed no significant adverse effects after weaning. 4. Discussion
Fig. 2. Score frequencies for behavioural development in the lactation period in two-generation toxicity study of tartrazine administered to mice. Significantly different from controls: *P < 0.05, ***P < 0.001. Surface righting at PND 4 in male offspring showed a significantly dose-related manner in a trend test (P < 0.01).
In the present study, tartrazine showed a few significant adverse effects on neurobehavioural parameters. The average body weight of offspring was significantly increased in the high-dosed group at PND 21 in male offspring. Nevertheless, the average body weights of male offspring in the high-dosed group were significantly increased at birth, and showed a tendency to be increased at PNDs 4, 7 and 14 (not significant). It therefore seems that the differences of male offspring weight between the control and high-dosed groups were caused not by tartrazine treatment but by the body size at birth. The survival indices showed a few significant adverse
Fig. 3. Movement activity of exploratory behaviour at three weeks of age of F1 generation male offspring administered tartrazine in the diet. Each value represents the mean ± SE. Movement activity of male offspring showed a significantly dose-related manner in a trend test (P < 0.05).
T. Tanaka / Food and Chemical Toxicology 44 (2006) 179–187
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Fig. 4. Summary of multiple-T water maze performance at seven weeks of age of F1 generation male mice in two-generation toxicity study of tartrazine. Each value represents the mean ± SE. Significantly different from the first trial: *P < 0.05.
effect in the middle-dose group during the lactation period. However, two litters of the middle-dose group died of accidents. It therefore seems that tartrazine produced no adverse effects on offspring survivals during the lactation period. In behavioural developmental parameters, tartrazine produced a few statistically significant effects in the measured variables as compared to control group in mice. Surface righting, indicative of the coordinated movement, was significantly accelerated in the high-dose group in male offspring in the early lactation period, and those effects were significantly dose-related (P < 0.01). Nevertheless, that of female offspring showed no significant effects during the lactation period. It therefore seems that tartrazine may have different effects on the behavioural development in sexes. In male offspring, number of movement of exploratory behaviour at three weeks of age in the F1 generation was significantly affected in a dose-related manner. Nevertheless, any variable measured showed no adverse
effects of tartrazine in movement activity of exploratory behaviour at eight weeks of age in the F1 generation in either sex. It therefore seems that tartrazine influenced exploratory behaviour in juvenile male mice only. In humans, Rowe and Rowe (1994) reported that some children who ingested tartrazine showed hyperactivity, and those effects showed dose-related (1–50 mg/child). Also, Ward (1997) reported that 23 children consumed a tartrazine beverage showed hyperactivity. Nevertheless, exploratory behaviour of mice was restrained by tartrazine treatment in juvenile males. It therefore seems that the dose levels of tartrazine in the present study may not induce hyperactivity in mice. From results, the high-dose level (0.45% in the diet) of tartrazine in the present study produced a few adverse effects on several behavioural parameters. Nevertheless, the high-dose level (645–2541 mg/kg/day) were in excess of the ADI of tartrazine (0–7.5 mg/kg bw). The actual dietary intake of tartrazine in Japan is presumed to be much lower, approximately 0.66 lg/kg/day in 1982
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Fig. 5. Summary of multiple-T water maze performance at seven weeks of age of F1 generation female mice in two-generation toxicity study of tartrazine. Each value represents the mean ± SE. Significantly different from the first trial: *P < 0.05.
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