Effects of amaranth on F1 generation mice

Toxicology Letters, 60 (1992) 3 15-324 0

1992 Elsevier Science Publishers

TOXLET

315

B.V. All rights reserved

0378-4274/92/$05.00

02708

Effects of amaranth on F, generation mice

Toyohito

Tanaka

Department qf Toxicology. Tokyo Metropolitan Research Laborurory of Public Health, Tokyo (Jupun) (Received

4 October

(Accepted

16 December

Key words; Amaranth;

1991) 1991) Reproductive

toxicity;

Developmental

toxicity;

Behavioral

toxicity;

Mice

SUMMARY The color additive,

amaranth,

was given in the diet to provide

dietary

levels of 0 (control),

0.03,0.09

and

0.27%, from 5 weeks of age in F, generation mice to 9 weeks of age in F, generation mice, and some reproductive, developmental and behavioral parameters were measured. There was no effect on the parameters

of litters,

period

in the treatment

litter size, pup weight groups

increased

and litter weight.

The body weight

of pups during

less significantly,

and the survival

index at postnatal

the lactation day (PND)

21 of the amaranth 0.27% group was reduced. Developmental parameters, direction of swimming on PND 4 in male pups and olfactory orientation in each sex were significantly reduced in the treatment groups. The dose levels of amaranth

in this study influenced

some reproductive,

developmental

and behavioral

parame-

ters in mice.

INTRODUCTION

The color additive, amaranth, is the trisodium salt of 1-(4-sulfo- 1-naphthyl-azo)-2naphthol-3,6-disulfonic acid. Amaranth is a red-brown, water-soluble powder widely used in food products, drugs and cosmetics. The standard level of amaranth as a food additive is from 5 to 100 ppm in the diet in Japan. The acceptable daily intake (ADI) is 0.5 mgikg body wt. [l]. Several reproductive toxicity studies of amaranth have been reported. Amaranth has shown no teratotogenic activity on mice [2] or in rats [335]. There has been no embryonic toxicity of amaranth [ 1,471. Since there have been few studies on the developmental and neurobehavioral toxicity of amaranth, this study was designed to evaluate its reproductive, developmental and neurobehavioral effects in mice over two generations. Correspondence fo; Toyohito tory of Public Health,

Tanaka,

Department

24-1, Hyakunincho

3 chome,

of Toxicology,

Tokyo

Metropolitan

Shinjuku-ku,

Tokyo

169, Japan.

Research

Labora-

316

METHODS

Amaranth Japan).

(Food

Red No. 2) was obtained

from Tokyo

Kasei Co., Ltd. (Tokyo,

Animals and maintenance Male and female mice (Crj:CD-1, 4 weeks old) were obtained from Charles River Japan Inc. (Tokyo, Japan). They were individually housed in polycarbonate solidfloored cages with wood flakes. They were given control or experimental diets and water ad libitum. E.upevimental design Amaranth was administered in the diet to 60 mice (IO/sex/group) at dietary levels of 0.03,0.09 and 0.27%. The 20 mice (lo/sex) in the control group were given the basal diet (Nihon Clea, CE-2) for a corresponding period of time. F,) generation The animals were 5 weeks old at the start of the study. At 8 weeks of age, the motor activity of the mice was measured by an animal movement analyzing system (ANIMATE AT-420, Toyo Sangyo Co., Ltd, Toyama, Japan). The apparatus consisted of a doughnut-shaped cage with detectors for measuring spontaneous motor activity. The behavioral parameters were recorded for 5 min. i.e. number of movements, movement time (s), number of horizontal movements, total distance (cm), number of vertical movements, vertical time (s), turning, average distance (cm), average speed (cm/s), and maximum continuation time (s) [8,9]. At 9 weeks of age, each female was paired with one male from the same treatment group, for a period of 5 days. After 5 days, the males were removed from the females, and the females were allowed to produce and rear their pups. F/ genevution On postnatal day 0 (PND 0), litter size, pup weight, and litter weight were measured. The pups were weighed on PND 4, 7, 14 and 21 during the lactation period. The physical and functional developmental parameters were measured for individual pups, and were analyzed on a whole-litter basis as follows: (I) Surface righting (PND 4 and 7) The pups were placed on their backs on a smooth surface and the time required to right themselves to a position where all 4 limbs touched the surface was recorded with a stopwatch. The scoring procedure for successful righting was: 2 = righting within 1 s; 1 = > 1 s but within 2 s; 0 = >2 s [lO,l 11. (2) Negative geotuxis (PND 4 and 7) The pups 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 with a stopwatch. The plane was made of plywood covered with sandpaper (fine grade). The following scoring procedure was employed: 0 = no response within 60 s; 1 = response within 60 s; 2 = response within 30 s [lo-121.

317

(3) Cliff avoidance (PND 7) The pups were placed on a platform elevated 10 cm above a table top. The forelimbs and snout of the animal were positioned so that the edge of the platform passed just behind an imaginary line drawn between the eye orbits. The following scoring procedure was employed: 0 = no response within 20 s; 1 = avoided backwards within 20 s; 2 = avoided with turn [lo-121. (4) Swimming (PND 4, 14) The pups were placed in a tank with water temperature maintained at 23?1 “C and swimming behavior was rated for direction (straight = 3, circling = 2, floating = 1) and head angle (ears out of the water = 4, ears half out of the water = 3, nose and top of the head out of the water = 2, and unable to hold the head up = 1). Limb movement was rated as either 1 = all four limbs used, or 2 = back limbs only used [lO,ll]. (5) Olfuctory orientation (PND 14) The pups were placed in the arm of the apparatus constituted by two compartments connected with the arm. One compartment was covered with the home wood flakes and the other was covered with fresh flakes. The time required to enter the home compartment was recorded with a stop-

TABLE MOTOR

I ACTIVITY

OF F, GENERATION

MICE

ADMINISTERED

AMARANTH

AT 8 WEEKS

OF AGE Dose level (%):

10

No. of males examined No. of movements Movement time (s) No. of horizontal movements Total distance

(cm)

No. of vertical

movements

Vertical Turning

time (s)

Ave. distance

1203.5f53.33 268.2k5.13 22.5k3.19 1605.4k168.23 86.7k5.23 114.2k4.07 28.1?2.51 117.4k46.59

(cm)

0.03

0

0.09 IO

10 1190.3-t66.52

1179.2k27.87

IO 1142.9+48.09

268.725.58 23.Ok2.77

269.5k3.63 21.4k2.15

264.81-5.89 24.1-Ll.39

1618.3+195.32

1609.1k62.59

1493.8+135.16

75.7k3.97 104.9i4.09 26.2k3.28 90.5f21.48

82.2k4.14

77.4k5.34

114.7k6.33 3O.lk1.72

101.2-t6.46

86.2-tl3.53

Ave. speed (cm/s) Max. cont. time

5.90-t0.524

5.92k0.623

5.97+0.210

37.24k4.305

33.28k3.704

38.13t-3.538

No. of females examined No. of movements

10 911.5228.42

10 957.5k75.65

Movement time (s) No. of horizontal movements

25l.lk3.93 29.0-t I .42

10 908.3k47.18 248.9k8.93

Total distance (cm) No. of vertical movements Vertical Turning

time(s)

Ave. distance (cm) Ave. speed (cm/s)

70.7k4.03 84.9k6.28 25.8k2.43 50.814.41 5.6520.263 28.47k3.981

Max. cont. time (s) Each value represents

1423.5277.97

the incidence

0.27

25.6k3.52 1393.4-tl24.59

246.92 13.83 23.7k2.73

25.2k4.27 66.1k9.56 5.58k0.416 3 1.97k2.868 IO 976.9k73.78 253.1-tl4.29 24.5k3.04

1757.3f406.80

1679.5k271.18

84.8k8.64 25.3k1.69 75.2k18.56

72.7+4.00 72.8k4.38 25.5k3.78

69.8k6.17 73.lk7.18

155.5k96.79

5.58+0.415 29.81k5.470

6.71k1.269 33.92k4.648

100.5k33.87 6.39kO.798 34.56t-5.566

77.2k8.24

per 5 min (mean_tSE)

24.4k3.65

318

TABLE

II

SUMMARY OF DATA TERED TO MICE

OF LITTERS

Dose level (%):

0

No. of litters No. of pups Litter size Pup weight (g) Litter weight (g) Each value represents

TABLE

IN F, GENERATION

STUDY

OF AMARANTH

0.03

ADMINIS-

0.09

0.27

9

10

9

10

109

124

109

97

12.1+0.93

12.4k2.80

12.lk2.67

1.57+0.152 18.9621.23

I .54*0.135

1.54~0.150

19.12k3.79

18.68+3.50

9.lk4.55

I .54*0.172 14.95k6.33

the mean_tSD

III

PUP WEIGHT(g) RANTH

IN LACTATION

PERIOD

52 1.5910.169

PND4

45 2.98-tO.406

PND 7

45 4.50+0.483

MICE ADMINISTERED

0.03

0

Dose level (%): kf& plqs PND 0

IN F, GENERATION

0.09

AMA-

0.27

54 1.57+0.163

42 1.56&O. I32

44 3.06kO.447

32 3.03kO.394

48 4.2820.842

44

32

44

58 1.59kO.128 49 2.78+0.564

4.59-t0.687

4.68kO.703

PND 14

45 6.83+0.573

47

PND 21

45

47

7.17kO.894 44

11.14+1.906

I I .37+2.010

12.07+- I.757

12.56k2.029

57

66

55

1.5O-tO.124 48

I .52?0.135 49 2.91+0.364 49

55 1.53-tO.198

Fmtrk pup.c PND 0

I .54+0. I32 PND 4

53 2.961-0.435

PND 7

53 4.50+-0.622

6.78-tl.017

2.72f0.487 48

14

53 6.70+0.840

4. I 1+0.741 48 6.70+0.989

PND 21

53 10.81+1.698

48 10.88i1.836

PND

Each value represents

number

of pups and the mean+SD

4.38-t0.564 49 6.93kO.718 49 11.33+1.173

32 7.45+ 1.040 32

39 3.08+0.538 39 4.7720.944 39 7.73+ 1.578 39 12.58k2.349

319

watch. The following scoring procedure was employed: 0 = no response within 90 s; 1 = entered the home compartment via the fresh compartment; 2 = entered the home compartment directly [12-l 41. At 3 weeks of age, the motor activity of the pups was measured, both males and females from each litter, in the same way as for the F, generation. At 4 weeks of age, the pups were removed from their dams, and were selected to continue treatment, one male and one female from each litter. At 7 weeks of age the mice performed 3 trials in a multiple water T-maze. Water

TABLE

IV

SUMMARY

OF BEHAVIORAL

AMARANTH

DEVELOPMENT

IN THE LACTATION

0

Dose level (%):

OF F, GENERATION

MICE ADMINISTERED

PERIOD 0.03

0.09

0.27

Mule pup Surface

righting

Negative

geotaxis

Cliff avoidance Swimming Direction

4

0.2a-to. 185

0.18-tO.195

0.32-tO.242

0.02-tO.O45*

7

1.69+0.170

1.4OkO.588

I .49?0.514

1.51+0.586

1.77kO.365 1.7820.667

1.93kO.095 2

1.93+0.163

1.5350.276

1.34kO.538

1.41 kO.338

1.90+0.113 1.48-tO.3 19

2.5420.263 3.2020.263 1

2.15+0.371* 2.8820.342

2.10+0.112**

2.09t-0.143*

2.93kO.406 1

3.00?0.184

4 7 I 4

Head angle Limb movement Swimming

2 3

1

Direction

3

3

Head angle Limb movement

4

4

Olfactory

1

14

orientation

2 1.54+0.253

14

3 4

3 4

2

2

2

1.16?0.288*

1.16-tO.318*

0.73?0.432**

0. lo-to. 174 1.34kO.626 1.81+0.414 2

0.14kO.212 1.64f0.282 2 2

0.18-tO.204 1.3520.375

1.59kO.228

1.34kO.388

1.36+0.208

1.46t0.340

2.39k0.248

2.04&0.414* 2.94kO.306

2.13kO.253

2.23zk0.242

2.83-t0.362

2.91+0.107

Femuk pups Surface Negative

righting geotaxis

Cliff avoidance Swimming

4 7 4 7

0.27kO.299 1.68-tO.320 2

7 4

Direction Head angle Limb movement Swimming

3.001-O. 176

orientation

Each value represents

1.94+0.103 1.9OkO.253

1

1

1

1

3 4 2

3 4

3 4 2

3 4 2

14

Direction Head angle Limb movement Olfactory

2

14 the mean_tSD.

1.66+0.18 1 Significantly

2 1.36?0.196* different

1.24t-0.301*

from controls:

1.16f0.248**

*P
320

TABLE

V

MOTOR

ACTIVITY

IN F, GENERATION

MICE

ADMINISTERED

AMARANTH

AT 3 WEEKS

OF AGE Dose level (%):

8

No. of males examined No. of movements Movement time (s) No. of horizontal movements Total distance (cm) No. of vertical movements Vertical Turning

0

time (s)

706. I k65.05 226.Ok I I .24 26.3kl.60 1099.0+98.32

8 742.4k74.36

564.7k67.79

240.5k8.75

223. I-t 16.40

195.5+16.8X

28.3+2.37 1400.9+101.94

13.8k3.07

5 I .4-t4.52

57.4+ I I .02

2X.8*3. II 4.17+0.228

Movement

208.87tl5.31 26.6-t I .65 962.6t 159.07 58.9k7.86

5.8O-tO.348 25. I I k2.880

53.6k2.32

I .77

5.44kO.404 32.26+6.041

x

8 546.9k63.24 199.9+37.72

548.3f57. IO 197.32 13.06

28.5kl.95 965. I + 134.40

27.4f2.56 919.9-t91.04

50.5k7.78

42.91-5.34

40.6f5.59

Turning

17.X-t2.89

Ave. distance (cm) Ave. speed (cm/s)

36.626.02 4.48k0.433

13.9t2.39 33.0+3.08

Max. cont. time (s)

26.40+4.X86 the incidence

29.Ok2.34 828.0-t 107.5 I

17.8+1.75

8 594.9k56.55

I

1246.4-t 164.74 42.9+

No. of females examined No. of movements

Each value represents

24.452.56

6

54.524.3

4.79-to.235

No. of vertical movements Vertical time (s)

8 774.6k44.22

45.6k5.40 22.9kl.94

24.981-3.292

time (s)

0.27

56. I k7.47

Max. cont. time (s)

No. of horizontal movements Total distance (cm)

0.09

44.355.38 21.3+2.9X 44.255.93

Ave. distance (cm) Ave. speed (cm/s)

0.03

4.69-t0.288 20.50+

I .746

46.0k7.93 36.6k7.28

49.Ok6.75 45.X+8.29

22.3324.980 7 624.0261.35 218.O-tl3.56 29.3-t2.18 999.2-t 107.42 54.1k7.65 42.9k5.31

16.0+2.45

18.4k3.64

35.1+4.19

35.2k4.66 4.5 I kO.255

4.6lkO.239 23.85+ I .405

21 .XO-t2.47X

of per 5 min (mean&SE).

temperature was maintained at 18&l “C. Time taken and number of errors were measured from start to goal up till 120 s. If the time taken was > 120 s, it was recorded as 120 s [15]. At 8 weeks of age. motor activity was measured in the same way as for the F,, generation. Statistical anulysis Student’s t-test was performed on litter size, pup weight, behavioral parameters were assessed with the Mann-Whitney

and litter weight. U-test.

The

RESULTS

F,) generation There was no consistent significant compoundor dose-related effect in motor activity of mice administered amaranth (Table I). However, there were reductions in some parameters in the high-dose group of males and in vertical time in medium- and

321

TABLE

VI

MULTIPLE

WATER

AMARANTH

T-MAZE

AT 7 WEEKS

PERFORMANCE

Dose level (%): No. of males examined Errors (no.) 1st day 2nd day 3rd day Time taken (s) 1st day 2nd day 3rd day No. of females examined Errors

OF F, GENERATION

MICE

ADMINISTERED

OF AGE 0

0.03

0.09

8

8

8

-

0.27 6

3.4+ 1.oo

8.1+1.93

4.3k1.16

4.3+1.15

4.1k1.32

5.8k1.61

3.5k1.30

2.2kO.70

3.lk1.32

4.5zbl.66

3.5? 1.24

4.5k1.28

57.8k14.34 47.9+ 12.08

88.62 15.83 57.1k12.94

35.1+12.57

37.Ok9.90

8

8

63.5k9.56 37.029.87 30.9k4.40 8

60.7?11.78 24.7k4.76 37.Ok5.86 7

(no.) 3.6tl.00 2.8kO.67

4.31-1.10

4.3-tl.10

3.450.95

2nd day

4.6+1.50

4.3k2.02

2.6kO.57

3rd day

3.3t1.19

3.5*1.00

2.1f0.44

2.720.78

75.3k9.65

77.9k13.21 47.1-tlO.05

68.3+11.81 51.5*15.71

74.1+13.60 38.4k7.31

33.527.72

29.5-tl3.05

27.6k5.55

1st day

Time taken (s) 1st day 2nd day 3rd day Each value represents

34. I k6.85 35.4+ 12.36 the mean+SE

high-dose groups of females. There were increases in several parameters in the medium- and high-dose groups of females, i.e. number of movement, average distance and average speed. F, generation

There were no consistent significant differences in the parameters of the litters (Table II). In the high-dose group, there were a few litters of small size. The body weight of the pups in the treatment groups during the lactation period was not significantly different from that of controls (Table III). However, there were increases in the body weight of the pups at PND 14-21 in the medium- and high-dose groups in both sexes. Several litters were annihilated due to underdeveloped mammary glands in the lactation period: 1 litter in the control and medium-dose groups, 2 litters in the low-dose group, and 3 litters in the high-dose group. The survival index at PND 21 for male pups was control 88.9%, low-dose 76.0%, medium-dose 82.5%, and highdose 7.5.0%, and for female pups was control 88.9%, low-dose 74.8%, medium-dose 88.9%, and high-dose 68.3%. As regards the developmental parameters, direction of swimming on PND 4 in males was significantly reduced in the treatment groups (Table IV). Olfactory orienta-

322

TABLE MOTOR

VII ACTIVITY

OF F, GENERATION

MICE

ADMINISTERED

AMARANTH

AT 8 WEEKS

OF AGE Dose level (%):

0

0.03

8

No. of males examined

8

No. of movements

120.3-t49.08

1240.9+63.52

Movement time (s) No. of horizontal movements

266.4k4.86 24.6+2.X6

270.0+4.00 19.6+3.61

1556.4-tl48.32

1955.X+297.60

Total distance (cm) No. of vertical movements Vertical

time (s)

x0.0+4.82 103.415.53

Turning

25.Ok3.77

Ave. distance

(cm)

74.X+15.61 5.7XkO.452

Ave. speed (cm/s)

79.3k4.17 103.6k6.94

Movement time (s) No. of horizontal movements

254. I k6.99 27.4f2.52

261 .O-c6.74 26.3k3.74

1459.5+182.81 77.4k5.70

1622.5-t174.80 x5.3+5.30

Ave. speed (cm/s)

5.64-tO.551 32.29c4.074

Max. cont. time (s) Each value represents

the incidence

I7 I7.6t 160.76

94.1+11.31

7. I5-tO.990

X 1015.9+55.95

26.6k2.87 63.7+- 16.52

I

30.1+3.41

8

(cm)

1783.4-t92.3

275.Ok3.68 2 I .5-t2.94

146.7k43.82

955.6236.61

Turning Ave. distance

274.8+1.71 20.02 I .32

x3.5*5.93

No. of females examined No. of movements

75.4+5.6X

6 1258.2+51.71

107.0+5.69 3 I .2-t2.52

40.x4+4.552

time (s)

x 1233.4k22.90

I I I .6?5.30 31.9+_3.10

38.lO-t5.720

Vertical

0.27

X5.6*2.69

Max. cont. time (s)

Total distance (cm) No. of vertical movements

0.09

6.50+0.309

96.Ok24.05 6.20+0.499

33.29+2.02X

39.0525.547

8

7

X95.5*46.50 245.9-i- 12.49

979.6*45.6X 26l.6k6.3 I

28.5k2.60 1306.45 139.6 I

25.123.51 1555.0+19X.x4

77. I k6.9 I

7X.426. I3 8 I .9-tX.59

X7.0+-4.69 24.5k3.15

X0.1+8.83 24.32 I .8X

73.2+13.16

51.81-10.53

6.141-0.566

.5.23+0.369 25.56k2.893

33.5Ok3.167

27.9t3.79 82.0-t25.66 5.87iO.649 33.01 t-4. I73

of per 5 min (mean?SE).

tion in both sexes showed a significant tendency to be repressed in the treatment groups. Other parameters of development showed no consistent significant compound- or dose-related effects. There was no significant difference in motor activity at 3 weeks of age, but some parameters in males showed interesting changes (Table V): there were increases in the low- and medium-dose groups, and a reduction in the high-dose group as regards number of movements, total distance, average distance, and average speed. No significant effects were found in the multiple water T-maze test (Table VI) or in motor activity at 8 weeks of age (Table VII), but there was an increase in turning in males in the treatment groups. DISCUSSION

There were a few biologically significant effects on reproductive, developmental and behavioral parameters in this study. In the movement activity test, there was no

323

consistent significant compoundor dose-related effect, but some parameters did show a tendency to be reduced or increased in the treatment groups. These effects may not have been clear because of the small sample sizes used, particularly in the F, generation. In the parameters of the litters, it might have been due to amaranth that there were a few litters of small litter size in the high-dosed group. In the developmental parameters, the direction of swimming in males was significantly reduced in the treatment groups at PND 4, but there was no significant effect at PND 14. It is suggested, therefore, that this parameter was reduced in the early period and recovered thereafter. Olfactory orientation was repressed in the treatment groups while the body weight of the pups at PND 14 was increased less significantly in comparison with the controls. It is suggested that this effect was caused not by growth repression but by underdevelopment of the olfactory system. The dose levels of amaranth in this study produced adverse effects on some developmental and behavioral parameters. In particular, the developmental parameters were repressed in the amaranth 0.03% group (about 50 mgikgld). This dose level was introduced on the basis of AD1 (0.5 mg/kg body wt.). Therefore, the developmental effects of amaranth should be examined with regard to the no-observed-effect level. REFERENCES 1 Clode, S.A., Hooson, J., Grant, D. and Butler, W.H. (1987) Long-term toxicity rats using animals exposured in utero. Food Chcm. Toxicol. 25, 9377946. 2 Larsson. K.S. (1975) A teratologic 4. 75582.

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