Toxicity and carcinogenicity studies of Caramel Colour IV in F344 rats and B6C3F1 mice

Fd Chem. Toxic. Vol. 30, No. 5, pp. 431-443, 1992

0278-6915/92 $5.00 + 0.00 Pergamon Press Lid

Printed in Great Britain

TOXICITY AND CARCINOGENICITY STUDIES OF CARAMEL COLOUR IV IN F344 RATS AND B6C3FI MICE K. M. MACKENZIE*,B. G. BOYSEN,W. E. FIELD and S. R. W. PETSEL Hazleton Laboratories America, Inc., PO Box 7545, Madison, WI 53707, USA C. I. CHAPPEL 1196 Botany Hill, Oakville, Ontario L6J 6J5, Canada J. L. EMERSON The Coca-Cola Company, PO Drawer 1734, Atlanta, GA 30301 and J. STANLEY PepsiCo Research and Technical Services, 100 Stevens Avenue, Valhalla, NY 10595, USA Aima'act--Caramel Colour IV, a type of caramel colour used in the manufacture of cola soft drinks, was evaluated for subchronic and chronic toxicity in rats, and carcinogenicity in Fischer-344 (F344) rats and B6C3F~ mice. In each of the studies, Caramel Colour IV was mixed with demineralized water and the solutions given to the animals ad lib. in the drinking fluid. The concentrations of Caramel Colour IV in the drinking fluid were adjusted periodically to achieve the desired caramel colour intake per kg body weight. In the range-finding studies, groups of 30 rats/sex were given Caramel Colour IV at levels of 0, 15, 20, 25 or 30 g/kg for 13wk, and groups of 10 male rats were given levels of 0, 2.5, 5, I0 or 15 g/kg for 6 wk followed, for some dose groups, by a 2-wk withdrawal period, and then re-initiation of dosing for another 2 wk. In the rat chronic toxicity study, levels of Caramel Colour IV of 0, 2.5, 5, 7.5 or 10 g/kg were given to groups of 25 rats/sex for 12 months. The test groups in the rat and mouse carcinogenicity studies were composed of 50 animals/sex and each species was given the caramel colour at levels of 0, 0, 2.5, 5 or 10 g/kg for 24 months. In each of the studies, treated animals tended to have dose-related lower water consumption than controls. This was attributed to poor palatability of the drinking fluid, and was generally associated with decreased food consumption and body weights. Rats given caramel colour often had soft or liquid malodorous faeces although there were no treatment-related ante-mortem observations in mice. Blood biochemical changes in the rat (i.e. reduced blood urea nitrogen, alkaline phosphatase and total serum protein) appeared to be related to dietary influences and were not considered toxicologically significant. There were no treatment-related alterations in haematological variables or treatment-related differences in survival or in the incidence of benign or malignant tumours among treated and control groups and no toxicologically important pathological findings. On the basis of these studies, Caramel Colour IV was not toxic or carcinogenic in F344 rats or B6C3F, mice. The highest dose level tested in the long-term studies (I0 g/kg) was considered to be the no-observed-adverse-effect level (NOAEL).

INTRODUCTION Caramel colours have recently been characterized (Licht et al., 1992a,b) and the specifications have been described (Licht et al., 1992c). Caramel Colour IV is characterized by having a range of total nitrogen content between 0.3 and 1.7%, and a range of total sulphur content between 0,8 and 2.5%, per 0.1 units absorbance at 610nm (Licht, 1992a). It is known as soft drink caramel and is widely used in such beverages throughout the world. Within this class of caramel colour, the colour intensity at 610 nm (K6~o) can range from 0.10 to 0.60 (Licht, 1992c). The samples used for these studies had a colour intensity

of 0.I (/Qio). *Present address: RMT, Inc., PO Box 8923, Madison, WI 53708-8923, USA. Abbreviations: BUN = blood urea nitrogen. NOAEL = noobserved-adverse-effect level. 431

A range-finding toxicity study of this caramel colour in the mouse has been reported (Tierney, 1979). Groups of 10 B6C3F l weanling mice per sex were fed levels of 0, 10, 15, 20 and 30% Caramel Colour IV in the drinking water for 4 wk. As based on fluid consumption data and average body weights, these concentrations o f caramel colour were equivalent to daily doses of 20-22, 32-40, 44-50 and 63 g/kg body weight/day. Fluid consumption was depressed throughout the study among all treatment groups. These changes, however, were not consistent with time or dosage. F o o d consumption was not statistically significantly different between treatment groups and controls. Body weight gains at 28 days were significantly depressed at the highest dose level in males and there were transient depressions in body weight gain at this dose level in females. Body weight gain was not affected at lower dose levels in either male or female mice. At autopsy the only treatment-

432

K.M. MAcKeNzIE et al.

related effect reported was enlargement of the caecum. A range-finding toxicity study in utero of Caramel Colour IV has also been conducted (Tierney, 1980). Groups of 12 young mature F344 rats per sex were fed levels of 0, 10, 15, 20 and 25% of this caramel colour in the drinking water for 21 days before mating, throughout pregnancy and lactation and for 90 days post-weaning to the progeny. On the basis of body weight data and fluid consumption measurements, these concentrations in the drinking water were equivalent to ranges of caramel colour intakes of 10-12, 15-16, 21-22 and 26-31 g/kg body weight/day, respectively, to the F0 generation before mating and 7-27, 11-42, 16-56 and 23-100 g/kg body weight/day, respectively to the F~ progeny from weanling to 90 days of age. There were no deaths in the parent generation. A dose-related decrease in body weight gain was noted in both the parent generation and progeny. Maternal weight gain was reduced at the 15, 20 and 25% concentrations and this was associated at these dose levels with reduced pup weight at birth. Litters were smaller in groups given the 25% concentration in drinking water. Haematological and clinical chemistry studies at 45 and 90 days of the progeny given caramel colour did not reveal significant differences from controls. Autopsy of the progeny at the end of the 90-day treatment period showed increased absolute and relative organ weights for the liver, kidney and caecum at dose levels of 15% and higher with brownish discoloration of the mucous membranes of the lower bowel and mesenteric lymph nodes. The 10% concentration of Caramel Colour IV in the drinking water was considered to be a no-effect level for both the parent generation and progeny. The objective of the studies described in this paper was to determine the effects of short-term (6- and 13-wk) and prolonged (1- and 2-yr) oral administration of high levels of Caramel Colour IV on the F344 rat and effects of prolonged (2 yr) oral administration on the B6C3F~ mouse. Caramel Colour IV was given to the animals in their drinking water since beverages are the major route of human consumption. MATERIALS AND METHODS

Test material. Caramel Colour IV (also referred to as caramel colour) was manufactured by D. D. Williamson and Company, Inc. (Louisville, KY, USA). Batch No. N8P15 was used for the 6-wk range-finding and the 13-wk toxicity studies and Batch No. 2-115-C18 was used for the chronic toxicity and carcinogenicity studies. These batches of caramel colour conform to the International Technical Caramel Association specifications for Caramel Colour IV (Licht, 1992c). Analytical data for the test materials were as shown in Table 1.

Table 1. Analysis of samples of Caramel Colour IV Batch no. Analytical data

N8PI5

Colour intensity, Kr~0* Solids (%) 4-Methylimidazole (mg/kg) Total nitrogen (%) Total sulphur (%) Ammoniacal nitrogen (%) Sulphur dioxide (%)

0.14 69.8 165 0.63 1.08 0.02 0.04

2-115-C18 0.13 70.6 110 0.54 1.12 0.03 0.03

*Absorbance at 610nm, path length 1 cm, of 1 g caramel colour solids/litre of water.

The test materials were mixed at appropriate levels with water that had been partly demineralized by reverse osmosis and sterilized by ultraviolet light. The dose levels of caramel colour were expressed as g/kg body weight/day (g/kg) and concentrations in the drinking fluid were adjusted throughout the study to maintain the appropriate caramel colour intake based on body weight. Test solutions were analysed periodically throughout the studies for confirmation of test material concentration. Animals. Male and female F344 rats (28 days old) and male and female B6C3F~ mice (21 days old) were obtained from the Charles River Laboratories, Inc. (Wilmington, MA, USA). Each animal was assigned an identification number that accompanied all data from that animal throughout the study. Rats were identified with a numbered metal ear tag, and mice with a numbered metal neck tag. Housing and maintenance. All animals were housed individually in polycarbonate cages with hardwood bedding. NIH07 Open Formula Rat and Mouse Diet, manufactured by Zeigler Brothers (Gardner, PA, USA) or Teklad Test Diets (Winfield, IA, USA) was available ad lib. Appropriate test solutions were administered ad lib. from clear glass bottles fitted with stainless steel sipper tubes and ball valves. The animals were kept in temperature- and humiditycontrolled rooms at 72_+ 3°F, 50_+20% relative humidity, on a 12-hr light/12-hr dark cycle and acclimatized to the laboratory for a minimum of 2 wk before feeding caramel colour. 13-wk range-finding study in rats. 300 rats were assigned at random to five groups (30 rats/sex/group) that received 0, 15, 20, 25 or 30 g/kg for at least 13 wk. All rats were observed twice daily and moribund or dead animals noted; rats were examined at least once each week for signs of toxicity. Body weights and food consumption were recorded weekly, and fluid consumption daily. After 6wk on test, 10 animals/sex/group were selected at random for haematology, blood chemistry and urinalysis tests (Table 2); they were then killed and subjected to autopsy. Clinical pathology tests (Table 2) were also done on all surviving animals before termination of the study after 13 wk on test. Ophthalmic examinations were done before the interim autopsy and before termination of the study.

Toxicity studies of Caramel Colour IV Table 2. Clinical pathology tests conducted in 13-wk range-finding and chronic toxicity rat studies with Caramel Colour IV Haematology Haematocrit Reticulocyte counts* Haemoglobin White blood cell counts, Red blood cell counts total and differential Platelet counts* Prothrombin time Nucleated RBC counts* Blood chemistry Blood urea nirogen Albumin Glucose Globulin Alanine aminotransferase Albumin/globulin ratio* Aspartate aminotransferase Sodium Total bilirubin Cholesterol* Alkaline phosphatase Potassium Creatinine Chloride$ Total protein Serum proteins by electrophoresis* Urlaalysis Volume* Bilirubin Physical appearance Blood pH Urobilinogen* Protein Specific gravity Glucose Microscopic examination of sediment Ketones Orotic acid (9 months only)* *For rats in chronic (l-yr) toxicity study only. $For rats in 13-wk range-finding study only.

All animals were examined macroscopically post mortem. Selected organs were weighed. Tissues were collected, preserved in 10% phosphate buffered formalin, and examined microscopically (Table 3). 6-wk range-finding study in male rats. In order to define more exactly the tolerance of rats for Caramel Colour IV, a second range-finding study was conducted in male rats. 50 male rats were assigned at random to five groups (10 rats/group) that received 0, 2.5, 5.0, 10.0 or 15.0 g/kg for 43 days. The fate of each group thereafter was determined on the basis of the results of clinical pathology tests: the group on 2.5 g/kg was killed and discarded on day 47; the groups on 5.0 and 10.0 g/kg were killed and discarded on day 56. The group on 15.0 g/kg was treated for 43 Table 3. Organs and tissues evaluated microscopically in rat and mouse studies with Caramel Colour IV* Adrenals?

Ovaries?

Brain

Pancreas

Bone (vertebra) Caecum?, ~: Colon~ Duodenum Epididymides Eyes (in Zenker's Solution) Gall bladder~ Gross lesions Heart? Ileum~/ Kidneys$ Liver:l: Lungs Lymph nodes (cervical, mescnteric~:, and thoracic) Mammary gland Oesophagus

Parathyroids Pituitary Prostate Salivary gland Sciatic nerve Seminal vesicle Skeletal muscle Skin Spinal cord Spleen? Stomach Testest Thymusll Thyroid Trachea Urinary bladder Uterus

*Evaluated microscopically for control and high-dose rats in rangefinding and toxicity studies, and for all rats and mice in carcinogenicity studies. $Weighed for all rats. :l:Evaluated microscopically for all rats in chronic (l-yr) toxicity study. §Evaluated microscopically for mice in carcinogenicity study only. [[Weighed for rats in 13-wk range-finding study only.

433

days, fed basal diet for 13 days then again treated from day 56 until day 69, when the control and 15.0 g/kg groups were killed. All rats were observed twice daily for signs of morbidity and deaths, and examined at least once each week for signs of toxicity. Body weights were recorded before the study began and on days 2, 9, 13, 20, 24, 31, 34, 41, 44, 51, 54, 62 and 68. Food consumption was measured for days 1-11, 12-21, 22-32, 33-42, 43-49, 51-54, 56-62 and 63-68. Fluid consumption was determined daily during days 3-9, 14-20, 25-31, 35-41, 51-54, 57-62 and 65-68. Clinical pathology tests were done on all animals before the study started and on days 22, 33 and 43. Additionally, clinical pathology tests were done for the 5.0 and 10.0 g/kg groups on days 50 and 55, and for the 0 and 15.0 g/kg groups on days 12, 50, 55, 63 and 69. The clinical pathology tests included blood urea nitrogen (BUN) for all intervals (days 1, 12, 22, 33, 43, 50, 55, 63, 69), serum creatinine (days 1, 12, 22, 33, 43 and 69), serum total protein (days 1, 12, 22, 33 and 43) and serum ammonia (day 69). Urine collections took place over a 24-hr period on days 43, 50, 55 and 63. Volume and specific gravity were recorded, and on day 69 urine was analysed for urea nitrogen creatinine, ammoniacal nitrogen and total nitrogen. Chronic (l-yr) toxicity study in rats. 250 rats were assigned at random to five groups (25 rats/sex/group) that received 0, 2.5, 5.0, 7.5 or 10.0g caramel colour/kg body weight/day for 12 months. All rats were observed twice daily for signs of morbidity and deaths, and examined at least once each week for signs of toxicity. Body weights and food consumption were recorded weekly. Fluid consumption was recorded four times each week for the first 12 wk, and over a 48-hr period each week thereafter. Ophthalmic examinations took place before study initiation, at 6 months, and before study termination. Haematology, blood chemistry and urinalysis tests (Table 2) were done at 6 months and before study termination (12 months). In order to obtain more information on the low blood urea nitrogen values observed in the 13-wk study, urine was collected at 9 months specifically to determine urinary orotic acid levels. All animals were killed, subjected to autopsy and examined macroscopically. Selected organs were weighed. Tissues were collected, preserved in 10% phosphate buffered formalin, and examined microscopically (Table 3). Carcinogenicity (2-yr) study in rats. 500 rats were assigned at random to five groups (50 rats/sex/group) that received 0, 0, 2.5, 5.0 or 10.0 g/kg for at least 24 months. Caramel colour addition to the drinking water was started when the animals were 44 days of age. All rats were observed twice daily for signs of morbidity or deaths, and examined at least once each week for signs of toxicity or palpable tissue masses. Body weights and food consumption were recorded weekly. Fluid consumption was recorded four times

K.M. MAcKENzmet al.

434

each week for the first 12 wk, and over a 48-hr period each week thereafter. Ophthalmic examinations took place before the study began, then at 6, 12 and 18 months and subsequently before the study ended. All animals were killed, subjected to autopsy and examined macroscopically. Tissues were collected, preserved in 10% phosphate buffered formalin, and examined microscopically (Table 3). Carcinogenieity (2-yr) study in mice. 500 mice were assigned at random to five groups (nominally 50 mice/sex/group) that received 0, 0, 2.5, 5.0 or 10.0g/kg for at least 24 months. Caramel colour addition to the drinking water was started when the mice were 42 days of age. At this time it was noted that several animals had been wrongly sexed. After corrections were made and animals reassigned, the dosage groups (g/kg body weight) and numbers of animals were as follows: male 0 (51), 0 (50), 2.5 (49), 5,0 (52), 10.0 (50); female: 0 (49), 0 (50), 2.5 (51), 5.0 (48), 10.0 (50). All mice were observed twice daily for signs of morbidity or deaths, and examined at least once each week for signs of toxicity or palpable tissue masses. Body weights and food consumption were recorded weekly. Fluid consumption was recorded four times each week for the first 12 wk, and over a 48-hr period each week thereafter. All animals were killed, subjected to autopsy and examined macroscopically. Tissues were collected, preserved in 10% phosphate buffered formalin, and examined microscopically (Table 3). Data analysis. Where applicable, the data from each of the studies were analysed separately by sex. Body weights, body weight gains, feed consumption, fluid consumption, haematology, blood chemistry, urine volume with specific gravity and pH, organ weights, and ratios of organ weights to body weights, were analysed using the one-way ANOVA technique (Sokal and Rohlf, 1969a). Mean values for urine protein, glucose, ketones, bilirubin, blood and urobilinogen were analysed by chi-square contingency

table techniques (Sokal and Rohlf, 1969b). Group mean values for treated groups that differed significantly from controls by ANOVA (P < 0.05) were compared with the mean values for each of the control groups by Dunnett's t-test (Dunnett, 1964). Survival of treated groups was compared with each of the two control groups using the method described by Thomas et al. (1977). The incidence of congestion of the mesenteric lymph nodes among treated and control groups in the carcinogenicity study in mice was analysed by the Fisher-Irwin exact test for group comparisons (Thakur et al., 1985). RESULTS The results of these studies are presented in Tables 4-16. In order to summarize the large amount of data accumulated, clinical pathology and organ weight data that did not indicate treatment-related statistically significant differences between specific treatment and control groups for one or both sexes have been omitted from the tables.

13-wk range-finding study in rats Throughout this study, rats given caramel colour (15-30g/kg) had dark, soft or liquid malodorous faeces. There were no other clinical effects, no deaths (Table 4) and no ophthalmic findings. Rats given caramel colour had reduced body weight gains, and at the end of the experimental period mean body weights of all treated male groups and of female groups given 20, 25 or 30 g caramel colour/kg were significantly lower than those of controls (Table 5). Water consumption (i.e. fluid consumption corrected for caramel colour solids) and food consumption of all treated groups of both sexes were significantly lower than those of controls throughout the study (Table 5). After 6 wk on test, male and female rats given caramel colour had significantly lower BUN levels, and male (but not female) rats had significantly lower

Table 4. Percentagesurvivalat study terminationin rat and mousestudies with Caramel Colour IV Treatment group Males Study 13-wk range-finding study in rats* 6-wk range-finding study in male rats? Chronic (l-yr) toxicity study in rats~: Carcinogenicity study in rats~ Carcinogenicity study in micell

Females

1

2

3

4

5

I

2

3

4

5

100

100

100

100

100

100

100

100

100

100

IO0

lOO

loft

IO0

IO0

92

100

100

100

100

100

100

100

I00

100

68

68

66

64

66

84

86

84

92

82

65

70

75 69 71 = not applicable *Dose levels:0, 15, 20, 25 and 30g/kg body weight. ?Dose levels:0, 2.5, 5.0, 10.0 and 15.0g/kg body weight. ~Dose levels:0, 2.5, 5.0, 7.5 and 10.0g/kg body weight. §Dose levels:0, 0, 2.5, 5.0 and 10.0g/kg body weight. IIDose levels:0, 0, 2.5, 5.0 and 10.0g/kgbody weight.

67

79

68

71

72

-

-

.

.

.

.

.

.

Toxicity studies of Caramel Colour IV

435

Table 5. In-life data from a 13-wk range-finding study in rats with Caramel Colour IV Caramel colour (g/kg) Variable (g)

Sex

Week

0

15

20

25

30

Body weight

M

0 13 0 13 1 13 I 13 I 13 1 13

109 331 88 185 94 122 77 76 120 167 97 102

109 303* 91 180 81" 92* 71 62* 107" 122" 91" 76*

109 296* 89 172" 79* 86* 66* 57* 110" 125" 88* 76*

108 274* 90 169" 72* 77* 64* 57* 106" 120" 87* 80*

I10 282* 90 162" 67* 80* 59* 49* 101" 134" 83* 82*

F

Food consumption

M F

W a ~ r consumption

M F

*Significant at P ~<0.05.

serum alkaline phosphatase levels than controls (Table 6). At the end of the treatment period (l 3 wk), BUN, and serum total protein and alkaline phosphatase levels, were significantly lower than those of controls in all groups given caramel colour. Rats given caramel colour had significantly higher urine specific gravity and lower pH values than control rats. Urinary volumes were also observed to be markedly lower in the treated groups. There were no alterations in haematological values in any of the treated groups. At the end of the treatment period, mean weights of the caecum (full and empty) for male treated rats were significantly higher and mean weights of the thymus, spleen, heart and gonads were significantly lower than those of controls (Table 7). In female treated rats, mean weights of the caecum and kidneys were significantly higher and mean weights of the thymus, spleen, heart and brain were significantly lower than those of controls. When expressed as a

percentage of body weight, only caecal and kidney weights were significantly higher than those of controls in both sexes for treated rats, and thymus weights were significantly lower in the treated groups of both sexes. The relative weights of other organs in treated groups did not differ significantly from those of controls. No histopathological changes, however, were observed in the thymus, spleen, liver or kidney in any of the test groups. Treated rats had dark staining of the mucosa of the caecum and colon, distension of the caecum, pigment accumulation in the macrophages of mesenteric lymph nodes, and increases in the thickness of the muscularis and mucosa of the caecum. 6-wk range-finding study in male rats There were no significant differences in body weights between treated and control groups (Table 8) and no deaths (Table 4). Food consumption was significantly lower than that of controls for only one

Table 6. Clinical pathology data from a 13-wk range-finding study in rats with Caramel Colour IV Clinical pathology tests Blood chemistry

Week 6

Sex M

F

13

M

F

Dose (g/kg)

Blood urea nitrogen (mg/dl)

Total protein (g/dl)

0 15 20 25 30 0 15 20 25 30 0 15 20 25 30 0 15 20 25 30

17.4 9.6* 10.6" 9.4* 10.8" 13.9 10.2" 9.6* 10.0* 10.5" 16.9 10.4" 8.2* 8.5* 7.9* 15.9 11.4" 10.9" I 1.3" 9.8*

6.6 6.5 6.3 6.3 6.2 6.3 5.9 6.2 6.1 6.1 7.1 6.7* 6.8* 6.7* 6.9 7.0 6.7* 6.6* 6.5* 6.7*

*Significant at P ~<0.05. FCT 30/~-.-G

Urinalysis

Alkaline phosphatase (IU/litre) 136 107' 105" 102" 102" 85 76 74 75 69 76 65* 64* 62* 62* 50 41" 40* 41" 46*

Specific gravity

Protein (mg/dl)

pH

1.052 1.078" 1.078" 1.082" 1.076" 1.044 1.076" 1.075* 1.065" 1.085" 1.043 1.086" 1.086* 1.085* 1.080* 1.058 1.088" 1.088" 1,086" 1,085"

107 220 300* 222 260 62 150 167 147 214 57 137" I 13* 144" 93 27 84* 88* 118" 84*

6.7 6.3 6.0 6.0 5.8 6.6 6.6 6.1 5.9 6.1 7.3 6.0 6.0 6.1 5.8 7.1 6.1 5.9 5.9 5.8

K . M. MACKENZIE et al.

436

Table 7. Organ weight data from a 13-wk range-finding study in rats with Caramel Colour IV Caecum (full) Week 6

Sex M

F

13

M

F

Dose (g/kg)

Percentage of BW

g

0 15 20 25 30 0 15 20 25 30 0 15 20 25 30 0 15 20 25 30

4.75 7.23* 8.92* 10.1" 12.1" 3.78 4.09 5.09 6.09* 8.03* 4.56 9.22* 10.3" 12.4" 14.9" 3.37 5.50* 6.67* 7.32* 10.3"

2.028 3.034* 3.908* 4.267* 5,420* 2.547 2.880 3.430 4.242* 5.445* 1.471 3.149" 3.557* 4.540* 5.404* 1.908 3.135" 3.929* 4,432* 6.432*

Caecum (empty) g 1.18 1.37 1.73" 1.87" 1.85" 0.803 0.886 1.04" 1.18" 1.29" 1.58 1.89" 2.14" 2.38* 2.61" 1.06 1.29" 1.42" 1.42" 1.73*

Spleen Week 6

Sex M

F

13

M

F

Dose (g/kg)

g

0 15 20 25 30 0 15 20 25 30 0 15 20 25 30 0 15 20 25 30

Percentage of BW

0,531 0.512 0.451" 0,477* 0.457* 0.382 0.356 0.377 0.365 0.356 0.585 0.550 0.543 0.506* 0.514" 0.429 0,400 0.397* 0.407* 0.364*

0.228 0.215 0.197" 0.203* 0.206* 0.261 0.252 0.254 0.252 0,242 0.188 0.187 0.188 0,185 0,187 0,242 0,228 0.237 0.246 0,229

Percentage of BW 0.506 0.574 0.756* 0.788* 0.834* 0.549 0.628 0,703* 0,817" 0.876* 0.508 0.645* 0.737* 0.870* 0.951" 0.600 0.739* 0,846* 0,859* 1.093* Heart

g 0,818 0,839 0.781 0.794 0.768 0.576 0.552 0,573 0.555 0,533 0.965 0.859* 0.876* 0.809* 0.832* 0,639 0.593 0.587* 0.574* 0.535*

Kidney (left)

Kidney (right)

g

Percentage of BW

g

Percentage of BW

0.910 0.993 0.970 0.978 0.941 0,635 0.675 0,705 0,697 0,695 IA0 1.14 1.15 1.12 1.13 0.681 0.759* 0.744* 0.756* 0.725*

0.389 0.417" 0.423* 0.416" 0.423* 0.433 0.480 0.475 0.483 0.472 0.353 0.387* 0.397* 0.406* 0.413" 0.383 0.433* 0.444* 0.457* 0.457*

0.906 0.993 0.979 1.0 0.969 0.626 0.668 0.702 0.686 0.691 1,08 1,13 1.14 1.10 1.13 0.681 0.768* 0.749* 0.748* 0.719

0.388 0.417" 0.427* 0.426* 0.435* 0.427 0.475 0.473 0.475 0.469 0.346 0.385* 0.394* 0.402* 0.411" 0.384 0.438* 0.446* 0,452* 0.453*

Gonad--left

Percentage of BW 0.351 0.352 0.341 0,338 0,346 0.393 0.391 0.386 0.384 0.363* 0.311 0.293 0.303 0.295 0.303 0.360 0.339 0.350 0.347 0.337

Thymus

0.338 0.363 0.349 0.332 0.345 0.308 0.289 0.280 0.285 0.298 0.309 0.241" 0,241" 0~234" 0,235* 0,242 0.231 0.227 0,189" 0.185"

Gonad--right

g

Percentage of BW

1.37 1.39 1.36 1.39 1.32 0.056 0.046 0.053 0.049 0.049 1.54 1.48 1.48 1.43" 1.49 0,062 0.063 0.067 0.061 0.056

0.588 0.583 0.593 0.590 0.590 0.039 0.033 0.036 0.034 0.033 0.496 0,505 0.513 0.524* 0.544 0,035 0.036 0.040 0.037 0.035

Percentage of BW

g

0.145 0.152 0.152 0.142 0.156 0.210 0.205 0.186 0.198 0.203 0.099 0.082* 0.083* 0.085* 0.086* 0.137 0.131 0.134 0,115" 0.116'

Brain

g

Percentage of BW

g

Percentage of BW

1.34 1.37 1.34 1.36 1.27 0.055 0.046 0.049 0,047 0.048 1.52 1.45" 1.46" 1.40" 1.46" 0.062 0.058 0.058 0,054 0.056

0.576 0.576 0.584 0.579 0.567 0.038 0.032 0.033 0.033 0.033 0.491 0.497 0.505 0.512 0.531" 0,035 0.033 0.034 0.033 0.036

1.85 1.82 1.83 1.80 1.79 1.65 1.72 1.67 1.70 1.68 1.88 1.86 1.88 1.88 1.87 1.77 1.74 1.72" 1.72* 1.71"

0.795 0.767 0.800 0.770 0.810 1.132 1.222" 1.128 1.177 1.142 0.607 0.638* 0.652* 0.688* 0.682* 0.998 0.996 1.028 1.042" 1.078"

BW = body weight *Significant at P ~<0.05.

time period (days 63-68) at the 15 g/kg dose level (Table 8). Water consumption in the groups given 10 and 15 g/kg tended to be slightly lower than that of controls during the early portion of the study. Clinical laboratory tests showed a treatmentrelated decrease in BUN levels (Table 9). At the 15 g/kg treatment level, BUN values returned to the

normal range within 7 days of cessation of caramel colour treatment (i.e. by day 50), indicating that this change was fully reversible. Serum creatinine and total protein levels were not affected by caramel colour at any interval or treatment level. The mean plasma ammonia level for the group given 15 g caramel colour/kg did not differ significantly from

Table 8. In-life data from a 6-wk range-finding study in male rats with Caramel Colour IV Caramel colour (g/kg) Variable (g) Body weight

Food consumption

Water consumption

*Significant at P ~<0.05.

Days

0

2.5

5

l0

15

2 41 68 1-11 33-42 63-68 4

I11 185 216 130 108 67 17.3

110 193 -125 110 -16.3

110 190 -125 107 -16.1

Ill 186 -120 96 -14.8

I10 180 209 116 94 56* 15. l

41 68

I 1.7 11.8

12.0 --

11.7 --

10.6 --

11.4 12.1

Toxicity studies of Caramel Colour IV

437

Table 9. Clinical pathology data from a 6-wk range-finding study in male rats with Caramel Colour IV Blood chemistry Total protein (g/dl)

0.0 2.5 5.0 10.0 15.0 0.0 2.5

18.5 18.8 19.0 19.1 18.9 12.5 12.3

5.5 5.5 5.3 5.7 5.6 6.0 6.1

0.7 0.7 0.8 0.8 0.7 0.7

. . . . . ---

5.0

11.7

6.0

0.7

--

1.055

3.0

.

6.1 6.0

0.8 0.7

---

1.057 1.037 1.039 1.055 1.061" 1.040 1.060

2.4 5.1 3.3 2.1 2.3

. . . . .

3.4

.

2.1

.

1.058 1.064

2.7 2.6 2.8

. . .

Dose Day (g/kg) -I

43

50

Urinalysis

Blood urea nitrogen (mg/dl)

10.0 15.0 0.0 (5.0)~f (10.0) (15.0)

55

0.0 (5.0) (I0.0) (15.0)

63

0.0 15.0

69

0.0 15.0

9.5* 8.1"

Serum Plasma Urea creatinine ammonia Specific Volume nitrogen (mg/dl) ~mol/litre) gravity (ml) (mg/dl)

0.9

13.5

--

--

--

13.5 12.8 12.6 13.4

-----

-----

-----

12.9

--

--

--

13.1

--

--

--

12.7

--

--

--

15.7

--

--

--

--

--

--

6.2 6.1

0.8 0.7

9.2*

15.8 8.5*

69.4 78.0

. . . . .

. . . . . 1.053 1.046

1.058 1.052 1.042 1.046 1.024"

. . . . . 2.9 3.7

Ammoniacal nitrogen (pg/ml)

Creatinine (mg/dl) . . . . .

. . . . .

. .

. . .

. . . . . . .

.

. . . . . .

. . . . . .

. . . .

. . .

. . . . . .

Total nitrogen (g/dl)

. . . .

. . . .

3.2 . . . 6.6 . . . 3.4 2,704.6 120.9 12.3" 925.9* 53.6'

. . 865 1004

3.52 i.13"

- - = test not done *Significant at P ~<0.05. '['Parentheses indicate that treatment was discontinued.

that of the control group. When treatment of caramel c o l o u r w a s r e i n s t a t e d in t h e 15 g / k g g r o u p f r o m d a y 56 t o d a y 69, s i g n i f i c a n t l y i n c r e a s e d 2 4 - h r u r i n a r y v o l u m e , d e c r e a s e d u r i n e specific g r a v i t y , u r e a n i t r o gen, creatinine a n d total nitrogen were observed. The NOAEL

f o r t h i s s t u d y w a s 5.0 g / k g .

t r e a t e d w i t h 10 g c a r a m e l c o l o u r / k g h a d s i g n i f i c a n t l y l o w e r b o d y w e i g h t s t h a n t h e c o n t r o l s ( T a b l e 10). D o s e - r e l a t e d l o w e r e d f o o d c o n s u m p t i o n o c c u r r e d in treated animals, Water consumption was lower for treated

groups

than

for controls, except for

Chronic (l-yr) toxicity study in rats T h e r e w e r e n o t r e a t m e n t - r e l a t e d d e a t h s ( T a b l e 4) o r o p h t h a l m i c f i n d i n g s in t h i s s t u d y a n d , e x c e p t for s o f t f a e c e s in t h e m a l e g r o u p s , n o t r e a t m e n t - r d a t e d a n t e - m o r t e m observations. Males, but not females,

i n c l u d i n g c o n t r o l s ( T a b l e 10). A t 6 a n d 12 m o n t h s , B U N w a s s i g n i f i c a n t l y l o w e r in m a l e a n d f e m a l e r a t s g i v e n 5.0 g c a r a m e l c o l o u r / k g o r m o r e . S e r u m c r e a tinine decreased in m a l e (but n o t female) rats given 5.0 g / k g o r m o r e b u t a l l v a l u e s w e r e w i t h i n n o r m a l

Table 10. In-life data from a chronic (l-yr) toxicity study in rats with Caramel Colour IV C a r a m e l colour

(g/kg)

Variable

Sex

Week

0.0

2.5

5.0

7.5

10.0

Body weight (g)

M

0 13 26

118 313 346

116 317 349

116 311 344

113 307 337

118 298 327*

52 0

391 93

384 94

386 92

374 96

361" 96

13 26 52

171 182 214

176 184 217

174 185 217

176 184 213

172 182 207

95 105 91 95 72 66 59 72 36.0 46.8 41.9 40.9 26.8 36.6 33.8 36.0

91 101 89 93 71 68 59 70 36.1 44.8 40.9 37.6 30.2 39.4 37.0* 36.2

88* 99* 87 95 73 65 55 69 35.4 42.4* 40.3 38.1 32. I * 33.0 32.0 30.8*

85* 94* 82* 87 69 64 54* 67 32.8 42.5* 38.6 38.4 29.2 32.4* 30.4 30.8*

84* 86* 78* 84* 68* 61" 50* 64* 31.9" 40.5* 40.1 37.9 28.2 29.9* 28.5 29.3*

F

Food consumption (g)

M

I

13 26 52 F

Water consumption (g]48 hr)

M

F

*Significant at

P ~ 0.05.

the

f e m a l e g r o u p g i v e n 2.5 g / k g , w h i c h h a d h i g h e r m e a n w a t e r c o n s u m p t i o n t h a n all o t h e r female g r o u p s

1

13 26 52 I 13 26 52 1 13 26 52

438

K . M . MACKENZIE et al.

Table I 1. Clinical pathology data from a chronic (1-yr) toxicity study in rats with Caramel Colour IV Blood chemistry Blood urea Serum Urine Dose nitrogen creatinine specific Month Sex (g/kg) (mg/dl) (mg/dl) gravity 6 M 0.0 13.3 0.89 1.056 2.5 12.5 0.92 1.059 5.0 12.0* 0.81* 1.063 7.5 10.9* 0.81* 1.066 10.0 9.6* 0.79* 1.070* F 0.0 14.4 0.96 1.051 2.5 12.9 0.89 1.048 5.0 12.8 0.91 1.062 7.5 11.2* 0.89 1.061 10.0 10.4" 0.89 1.062 12 M 0.0 14.1 1.1 1.046 2.5 13.2 1.0 1.055 5.0 12.1" 0.9* 1.057 7.5 10.3" 1.0" 1.056 10.0 10.3" 1.0" 1.060" F 0.0 14.9 1.1 1.051 2.5 13.8 1.1 1.047 5.0 13.1" 1.1 1.048 7.5 12.4" 1.1 1.058 10.0 9.4* 1.1 1.056 *Significant at P ~<0.05. limits for the F344 rat. U r i n e specific gravity at 6 a n d 12 m o n t h s was slightly higher t h a n t h a t of controls for g r o u p s given 7.5 or 10g/kg; however, all means were within n o r m a l ranges (Table 11). There were n o o t h e r statistically significant changes in clinical chemistry a n d there were no haematological alterations d u r i n g the study. In spite of the changes in clinical chemistry values, there were no gross or microscopic pathological changes in the liver or the kidneys. C h a n g e s in o r g a n weights ( c o m p a r e d with those of controls) were limited to higher weights of the kidney a n d caecum; the differences were generally statistically significant in male and female rats given caramel colour at levels o f 5 g/kg or more (Table 12). There were no gross or microscopic pathological changes in the kidneys. Pigmentation, the severity of which was treatment related, was observed in the mucosa of the caecum and colon, a n d in macrophages of the mesenteric lymph nodes in rats given 5.0 g/kg or more. The pigmentation was n o t associated with reactive hyperplasia. The changes in various clinical a n d a n a t o m i c a l variables were n o t considered toxicologically i m p o r t a n t . The N O A E L for this study was 10.0 g/kg.

Carcinogenicity study in rats Except for dark, m a l o d o r o u s a n d soft faeces, there were no treatment-related a n t e - m o r t e m observations, o p h t h a l m i c findings, or differences in overall survival (Table 4). Rats treated with 5.0 or 10.0g/kg h a d significantly lower body weights (Figs 1 and 2) a n d water c o n s u m p t i o n t h a n did controls (Table 13). Dose-related lower m e a n food c o n s u m p t i o n was observed for treated rats. As in the 1-yr study, treatment-related effects were limited to the gastro-intestinal tract a n d associated l y m p h nodes. Macroscopic changes included d a r k b r o w n contents in the gastro-intestinal tract, and staining of the gastro-intestinal mucosae, perineum a n d mesenteric lymph nodes. U n f o r m e d contents were observed in the large intestine of treated animals. Pigment deposition t h a t appeared to be inert a n d caused no tissue reactions was observed microscopically in the s u b m u c o s a of the ileum, colon a n d caecum, and in m a c r o p h a g e s of the mesenteric lymph nodes in rats given 5.0 g caramel colour/kg or more. C h r o n i c progressive n e p h r o p a t h y and bile duct hyperplasia, frequently observed in F344 rats, were present at c o m p a r a b l e frequency in all of the test groups. All microscopic findings in this study, with the exception of gastro-intestinal a n d mesenteric lymph node pigmentation, were considered to be c o m m o n incidental findings in this strain of rats. C o m m o n l y occurring s p o n t a n e o u s neoplasms o f F344 rats were found in treated and control rats o f b o t h sexes. There were no significant differences in the incidence of either benign or m a l i g n a n t neoplasms between treated a n d control groups (Table 14) a n d no unusual or rare t u r n o u t s were observed. The N O A E L for Caramel Colour IV for this study was 10 g/kg body weight.

Carcinogenicity study in mice There were no treatment-related a n t e - m o r t e m observations or differences in overall survival (Table 4) and no consistent significant differences in m e a n body weights (Figs 3 and 4). A l t h o u g h food c o n s u m p tion was consistently depressed for males at 10.0 g/kg, there were no c o m p a r a b l e effects on body weights. Mice treated with 5.0 or 10.0g/kg generally

Table 12. Organ weight data from a chronic (I-yr) study in rats with Caramel Colour IV Caecum (full) Caecum (empty) Kidney (left) Kidney (right) Dose Percentage Percentage Percentage Percentage Week Sex (g/kg) g of BW g of BW g of BW g of BW 12 M 0.0 3.416 0.019 1.487 0.399 1.256 0.342 1.285 0.347 2.5 3.970 1.073 1.442 0.390 1.327 0.358 1.292 0.349 5.0 4.635* 1.242* 1.505 0.404 1.329 0.356 1.300 0.348 7.5 5.270* 1.447* 1.632 0.448 1.332 0.367* 1.296 0.357 10.0 6.391" 1.809" 1.761 0.499* 1.354" 0.382* 1.350 0.370 0.0 2.390 1.178 0.973 0,480 0.808 0.400 0.790 0.392 2.5 2.564 1.241 0.980 0,473 0.823 0.398 0,803 0.398 5.0 2,894 1.381 1.054 0.505 0.872* 0.417 0.838* 0.402 7.5 3.287* 1.604* 1.808 0.527 0.867* 0.424* 0,857* 0.419" 10.0 3.741' 1.857" 1.164" 0.580* 0.888* 0.443* 0.862* 0.430* BW = body weight *Significant at P ~<0.05.

Toxicity studies of Caramel Colour IV 500

•

+ Og/kg

Og/kg

•

02.5g/kg

439

5.0 g / k g

x 10.0g/kg

450

400

~

/t/p

350

m

E=

300

250

200

..x""

~

- * • " ---e" .. x ....

x. . . . . . . . .

x...

...x'~..... . x ..... x * . . * , x

150

100

50 0

I

I

I

I

I

I

I

I

I

I

I

I

I

8

16

24

32

40

48

56

64

72

80

88

96

104

W e e k on t e s t

Fig. 1. Mean body weights of male rats fed Caramel Colour IV for 104 wk in the drinking water. consumed less water than controls, but only the group given 10g/kg had significant differences (Table 15). 500

• Og/kg

+ Og/kg

Treatment-related gross and microscopic findings in this study were limited to the gastro-intestinal tract and mesenteric lymph nodes. At autopsy the ileum,

02,Sg/kg

•

5.0 g / k g

x 10.0g/kg

450

400

350

300

x

25O ~"'~x

200

•

•

d

.

~

"" x ' ""x'"'x

.....

............. x"'"x"'"

x""x

"*'"

_,~lt~_ .~--~.'.~. . . . . . . . . . ; ...................

-~'~t'''lt''

150

100

50 0

I

I

l

I

I

I

I

I

I

I

I

I

I

8

16

24

32

40

48

56

64

72

80

88

96

104

W e e k on t e s t

Fig. 2. Mean body weights of female rats fed Caramel Colour IV for 104wk in the drinking water.

44O

K. M. MACKENZIE et al. Table 13. In-life data from a carcinogenicity (2-yr) study in rats with Caramel Colour IV Caramel colour (g/kg) Variable

Sex

Week

0.0

0.0

2.5

Body weight (g)

M

0 13 26 52 78 104 0 13 26 52 78 104

116 302 358 406 446 430 92 172 197 230 276 302 92 101 104 105 119 II1 71 71 71 74 89 86 32.8 41.4 40.8 40.8 47.5 53.7 29.6 35.2 34.6 33.0 39.2 40.5

114 292 350 401 439 423 93 170 198 229 280 308 92 96 105 101 116 108 73 70 71 72 86 89 34.9 39.2 41.6 41.5 48.2 45.6 32.5 34.6 34.1 34.2 40.1 41.3

116 293 352 404 446 416 91 170 194 228 275 304 90 94* 99*'** 97* I 13* 101" 71 69 69*'** 71" 84* 85 35.0 39.4 39.7 40.1 48.7 46.6* 30.5 35.7 37.0** 32.9 38.9 38.4

Food consumption (g)

M

I

13 26 52 78 104 1

Water consumption

M

(g/48 hr)

13 26 52 78 104 I 13 26 52 78 104 1 13 26 52 78 104

5.0

10.0

111" 295 346 389* 424* 397*'** 91 169 194 227 269** 290**

113 288 341" 377*'** 407*'** 382*'** 91

87"**

85*'**

90*'** 96"** 94*'** 107"** 96"** 70 67*'** 65*'** 70* 82"** 80*'** 32.0** 38.3* 40.1 39.4** 45.2*'** 47.2 29.1"* 33.7 32.2* 28.8"** 34.3"** 36.2*'**

165"**

186"** 210"** 245*'** 269"** 84*'** 91"** 87*'** 96*'** 85"** 65* 60*'** 59*'** 62*'** 71"** 69"** 32.5 37.5* 38.7** 38.5** 43.1"** 44.5* 26.8*'** 28.0"** 27.8*'** 25.4"** 32.0*'** 33.9*'**

*Significant at P ~<0.05 for first control group. **Significant at P ~<0.05 for second control group.

caecum and colon contained dark-coloured contents a n d the m u c o s a l s u r f a c e s o f these o r g a n s w e r e s t a i n e d b r o w n . T h e s e c h a n g e s w e r e m o s t a p p a r e n t at the d o s e levels o f 5.0 a n d 10.0 g / c a r a m e l c o l o u r / k g . T h e r e were n o t r e a t m e n t - r e l a t e d m i c r o s c o p i c c h a n g e s in these o r g a n s . Diffusely red m e s e n t e r i c l y m p h n o d e s at g r o s s a u t o p s y t h a t a p p e a r e d c o n g e s t e d o n m i c r o s c o p i c e x a m i n a t i o n were p r e s e n t in b o t h t r e a t e d a n d c o n t r o l mice t h a t died d u r i n g the s t u d y o r were killed at the e n d o f the study. T h e r e w e r e n o signific a n t differences in the incidence o f m e s e n t e r i c l y m p h node congestion a m o n g treated or control male mice t h a t w e r e killed at t e r m i n a t i o n o r a m o n g females t h a t died o r w e r e killed d u r i n g the study. A m o n g

the m a l e mice t h a t died o r were killed d u r i n g the s t u d y o r females killed at s t u d y t e r m i n a t i o n , the incidence o f m e s e n t e r i c l y m p h n o d e c o n g e s t i o n w a s significantly increased o n l y at the highest c a r a m e l c o l o u r d o s e c o m p a r e d w i t h either c o n t r o l group. A variety o f b e n i g n a n d m a l i g n a n t t u m o u r s typical o f t h o s e t h a t o c c u r s p o n t a n e o u s l y in the BeC3F ~ m o u s e were f o u n d in b o t h t r e a t e d a n d c o n t r o l test g r o u p s . T h e incidence o f these t u r n o u t s w a s n o t affected b y C a r a m e l C o l o u r I V at a n y o f the d o s e levels a n d n o rare o r u n u s u a l t u m o u r s were o b s e r v e d ( T a b l e 16). T h e N O A E L for this s t u d y w a s 10.0 g Caramel Colour IV/kg.

Table 14. Summary of benign and malignant neoplasms in rats that died, were killed as moribund or that were killed at the end of a 2-yr feeding study of Caramel Colour IV* Dose received by males (g/kg) Dose (g/ks)

0.0

0,0

2.5

5.0

10.0

Dose received by females (g/kg) 0.0

0.0

2.5

5.0

10.0

42 43

42 46

42 41

36 48

5

4

8

39 27 10

8 6 3

8 10 7

14 4 3

11 9 6

Rats killed at termination No. of animals Total benign neoplasms Total malignant neoplasms

No. of animals Total benign neoplasms Total malignant neoplasms

34 85

34 86

30 76

32 75

32 79

6 5 2 5 8 10 Rats killed when moribund or that died during test

16 28 12

*Includes multiple tumours per animal.

16 28 11

20 44 14

18 30 8

18 30 14

8 5 4

Toxicity studies of Caramel Colour IV 50

45

r

• 0 mg/kg

+ Omg/kg

<> 2.5 mg/kg

• 5.0 rng/kg

+

40

_+~.---,---.,

441

*-+

x 10.0 mg/kg

+

+-----+----4~--;~P<~,~

. 4 - - ~ ~ " ~

'

"--'~1:'~

/,J

35

~

30

J/"'

25

20

15 0

I 8

I 16

I 24

I 32

I 40

I 48

I 56

I 64

J 72

I 80

I 88

I 96

I

104

Week on test

Fig. 3. Mean body weights of male mice fed Caramel Colour IV for 104 wk in the drinking water. DISCUSSION A pattern of dose-related changes was noted in these studies that is typical of the responses of the rat

50

• 0 mg/kg

+ Omg/kg

to the feeding of high dose levels of any of the caramel colours. These changes include reduced fluid and food intake, reduced body weight gain, decreased urine volume and increased urine specific gravity,

O 2.5 mg/kg

• 5,0 m g / k g

x 10.0 m g / k g

45

40 . ~

f

~ o ~

/J~'j*~--l;-~__+.---L~"

35

. x..'" x ....

~ .............

;: .... • . . . . . . . . .

~;.J,~..~ ....

3o

25

20

f J 0

I 8

I 16

I 24

I 32

J 40

1 48

I 56

I 64

I 72

I 80

I 88

1 96

Week on test Fig. 4, Mean body weights o f female mice fed Caramel Colour I V for ;04 w k in the drinking water.

I 104

K. M. MACKENZIE et al.

442

Table 15. In-life data from a carcinogenicity (2-yr) study in mice with Caramel Colour 1V Caramel colour (g/kg) Variable

Sex

Week

0.0

0.0

2.5

5.0

10.0

Body weight (g)

M

0 13 26 52 78 104 0 13 26 52 78 104 1 13 26 52 78 104 1 13 26 52 78 104

22.1 30.5 36.5 38.7 40.5 37.9 18.8 26.4 31,3 36.8 40.7 40.8 31.2 32.8 35.5 31.8 33.2 31.8 30.6 31.8 36.6 32.9 35.8 40.1

22.0 30.2 36.2 40.6 41.7 39.6 18.8 26.7 30.9 35.4 38.0 38.7 30.9 31.7 32.5 32.5 33.0 34.2 29.6 31.0 35.2 31.8 33.8 37.3

21.9 29.5 35.5 38.6 39.8 38.6 19.0 26.9 31.5 36.4 41.3 41.9 32.6 31.2" 32,3* 31,4 32,4 31.7 30.2 31.2 32.8* 32.6 36.2 39.1

22.2 30.2 36.6 39, I 39,9 40,5 18.8 26.1 31.3 34.8 38.3 39.9 31.7 31.7 33.6 31.5 32.4 33.2 31. I 30.9 33.5* 30.8 33.4 38.2

I 13

11.7 I 1.2

I 1.4

10.8

I 1.2

26 52 78

10.7 12.7 12.8

10~9 10,5 12.2 13.1

10.7 10.6 12.3 11.9

10.6 11.0 11.4 11.6

21.8 29.4 35.6 37.7 40.0 40.0 18.9 25.9 29.7 33.9 36.3* 38. I 31.4 30.4*'** 32.4* 29.3*'** 31.3 31.1"* 41.9 31.3 34.4 31.8 35.2 38.3 10.5 10.6 10.0 10.4".** 11.0"*

104 1

14.0 11.0

14.6 11.1

12.6

12.5

13 26 52 78 104

11.2 11.4 12.1 12.8 13.5

11.3 11.1 10.8 I 1.6 12.7

10.9 10.8 11.0 10.9 I 1.4 11.5

10.8 11.0 11.1 10.6 10.7 12.4

F

Food consumption (g)

M

F

Water consumption (g/48 hr)

M

F

11.5"** 9.7*,**

11.1 10.5 10.3 10.5 11.5

*Significant at P ~<0.05 for first control group. **Significant at P ~<0.05 for second control group.

increased kidney weights, caecal enlargement and p i g m e n t a t i o n o f t h e m u c o s a o f t h e l o w e r digestive tract and mesenteric lymph glands. These changes are c o n s i d e r e d to be n o n - s p e c i f i c a n d a d a p t i v e . It is well k n o w n t h a t w h e n t h e w a t e r i n t a k e o f r o d e n t s is limited, f o o d i n t a k e will be p r o p o r t i o n a l l y r e d u c e d a n d t h e r e will be a r e d u c e d rate o f b o d y w e i g h t gain. R e d u c e d w a t e r i n t a k e also affects r e n a l function, reducing urinary output and increasing specific g r a v i t y . A c o m p e n s a t o r y h y p e r t r o p h y o f t h e k i d n e y , w i t h o u t p a t h o l o g i c a l c h a n g e s , also o c c u r s . T h e r e n a l h y p e r t r o p h y t h a t o c c u r r e d in this investig a t i o n m a y be a s s o c i a t e d w i t h d i s t u r b e d w a t e r bala n c e s e c o n d a r y to d e c r e a s e d w a t e r c o n s u m p t i o n , d e c r e a s e d u r i n e v o l u m e , a n d w a t e r loss in t h e faeces.

T h e s e a n d o t h e r a d a p t i v e c h a n g e s to t h e f e e d i n g o f h i g h c o n c e n t r a t i o n s o f c a r a m e l c o i o u r s in t h e d r i n k i n g w a t e r a r e d i s c u s s e d in g r e a t e r detail in M a c K e n z i e et aL (1992). B l o o d b i o c h e m i c a l c h a n g e s (i.e. r e d u c e d B U N , alkaline phosphatase, and total serum protein) were n o t e d in r a t s g i v e n h i g h d o s e s o f C a r a m e l C o l o u r IV, T h e s e c h a n g e s were n o t a s s o c i a t e d w i t h g r o s s or m i c r o s c o p i c p a t h o l o g i c a l c h a n g e s in t h e liver o r t h e k i d n e y s . T h e c a r a m e l c o l o u r u s e d in t h e s e s t u d i e s c o n t a i n s a n a p p r e c i a b l e level o f u n r e a c t e d g l u c o s e ( a p p r o x . 3 0 % ) ( L i c h t et al., 1992a), a n d d i f f e r e n c e s in B U N m a y be d u e to r e s i d u a l g l u c o s e levels. G l u c o s e a n d o t h e r s u g a r s h a v e b e e n s h o w n to r e d u c e B U N in a n i m a l s ( P o t e z n y et al., 1986; R o f e et al., 1986). T h e

Table 16. Summary of benign and malignant neoplasms in mice that died, were killed as morbund or that were killed at the end of a 2-yr feeding study of Caramel Colour IV Dosereceivedby males(g/kg) 0.0

0.0

2.5

5.0

10.0

Doser~eivedby ~males(g/kg) 0.0

0.0

2.5

5.0

I0.0

31 28 19

34 23 19

33 26 18

34 33 17

33 13 12

13 5 6

17 10 9

14 7 6

17 6 9

Mice killed attermination

No. of animals Total benign neoplasms Total malignant neoplasms

33 21 15

34 32 13

36 28 14

35 27 17

34 20 14

Mice killed when m o ~ b u n d o r that d i e d d u ~ n g t e s t

No. of animals Total benign neoplasms Total malignant neoplasms

18 7 14

16 14 13

12 10 8

17 8 12

15 4 13

18 5 11

Toxicity studies of Caramel Colour IV changes in the blood chemical variables may be related to dietary intake and are not considered toxicologically important. Caecal enlargement and pigmentation of the gastro-intestinal tract and mesenteric lymph nodes were noted in the studies of Caramel Colour IV. On the basis of results of studies in the rat and the mouse, Caramel Colour IV is free of specific toxicity even at extremely high feeding levels. Treatment with caramel colour in the drinking water at levels of up to 10.0 g/kg over a 2-yr period caused neither neoplastic nor non-neoplastic toxicological changes in F344 rats or B6C3Ft mice. The effects of caramel colour given in the drinking water on food and water intake, and indirectly on renal function, are nonspecific nutritional and physiological responses that reflect adaptation to the high concentrations that were fed.

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