Long-term feeding study on chocolate brown FB in rats

Fd Cosmet. Toxicol. Vol. 10, pp. 3-15. Pergamon Press 1972. Printed in Great Britain

Research Section Long-term Feeding Study on Chocolate Brown FB in Rats I. F. GAUNT and P. G. BRANTOM--ToxicoIogy P. GRASSO and MARGARETCREASEY Pathology and

S. D. GANGOLLI--Analytical Chemistry British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, England (Received 5 July 1971) Abstract--Groups of 30 male and 30 female weanling rats were fed for 2 yr on diets containing 0 (control), 1000, 3000, I0,000 or 30,000 ppm Chocolate Brown FB. No effects attributable to treatment were found in mortality, body-weight gain, haematology, serum chemistry, organ weights or incidence of tumours. There was pigment deposition in the renal tubular cells at dietary levels of 3000 ppm and above. Pigment was also found in the Kupffer cells of the liver and in cells of the lymph nodes, spleen and intestinal mucosa of a few female rats at the 30,000 ppm level. No carcinogenic potential was detected in Chocolate Brown FB and the no-untoward-effect level was 1000 ppm (approximately 50 mg/kg/day).

INTRODUCTION Chocolate Brown FB is listed in the 1956 Colour Index but has no number or specification. It is prepared by diazotizingnaphthionic acid and coupling with morin and maclurin (2',3,4',5,7-pentahydroxyflavone and pentahydroxybenzophenone). It is one o f the colourings at present included in the U K permitted list (The Colouring Matter in Food Regulations 1966, Statutory Instrument 1966, no. 1203). However, the reports o f the F o o d Standards Committee (1964) and the Joint F A O / W H O Expert Committee on Food Additives (1965) stressed the need for further evidence o f safety-in-use if Brown FB were to continue in use as a food colouring. Fore, Walker & Golberg (1967) reported that Brown FB was broken down in vitro by rat-liver homogenates but the rate was considerably less than that with Brown F K and Chocolate Brown HT. Gaunt, Hall, Farmer & Fairweather (1967) found that daily intubation of doses o f 2000 mg/kg in rats or I000 mg/kg in mice was tolerated for 3 wk without death. In a short-term feeding study, they established a no-effect level of 3000 ppm in the diet o f rats for 90 days (approximately equivalent to an intake of 150 mg/kg/day). At a level o f 10,000 ppm, pigment was found in the cells o f the alimentary canal, lymph nodes and renal tubules and it was also found in the Kupffer cells o f the liver at 30,000 ppm. This pigment was thought to represent an uptake of protein-bound colouring by the cells of the reticulo-endothelial system. 3

4

I . F . GAUNT, P. G. BRANTOM, P. GRASSO, MARGARET CREASEY a n d S. D. GANGOLLI

As a continuation of the investigation of the safety-in-use of Chocolate Brown FB, a 2-yr feeding study was included in the BIBRA safety evaluation programme. The findings are reported in this paper. EXPERIMENTAL

Materials. The sample of Brown FB used was supplied through the Food Colours Committee of the Chemical Industries Association. There is no British Standard Specification for Chocolate Brown FB, but the sample was stated to contain 81.8~ pure dye, 6~o volatile matter (loss at 135°C), 0.05~ water-soluble material, 0"9~o ether-extractable material, 11.3 ~o chlorides and sulphates (expressed as sodium salts), 0.5 ppm arsenic, 2.0 ppm lead and 14.0 ppm copper. Animals and diet. Both sexes of CFE rats obtained from an SPF colony (Carworth Inc., USA) were maintained on Spillers' Laboratory Small Animal Diet and water ad lib. They were housed in a room kept at a temperature of 20 A- I°C with a relative humidity of 40-50 ~o. Experimental design and conduct. Groups of 30 male (90-110 g body weight) and 30 female (80-100 g body weight) weanling rats, housed five per cage were fed diets containing either 0 (control), 1000, 3000, 10,000 or 30,000 ppm Chocolate Brown FB for 2 yr. Body weight and food consumption were recorded every 2 wk. Blood for haematological investigations was collected from the caudal veins of 12 rats of each sex from groups fed the control diet and the 30,000-ppm dietary level at wk 12 and from a similar number of animals of each sex from each of the groups at wk 28, 51 and 81. Blood was collected from the aorta of all surviving rats during autopsy after treatment for 104 wk. All the blood samples were examined for haemoglobin concentration, packed cell volume and erythrocyte and leucocyte counts. Reticulocytes, erythrocytes with Heinz bodies and the various types of leucocyte were counted in blood taken from the control, 10,000- and 30,000-ppm groups at wk 28-81 and in all samples taken at wk 104. Differential leucocyte counts were also made on the blood samples collected at wk 12. The methaemoglobin concentration was measured (Sunderman & Sunderman, 1960) in the samples taken at wk 104. Serum was separated from the blood collected at autopsy and measurements were made of the urea concentration and levels of glutamic-oxalacetic and glutamic-pyruvic transaminases. Urine analysis and kidney function tests were conducted on all rats surviving after 52 wk and on 12 rats from each sex from each group during the last 2 wk of feeding. The urine was examined for colour, microscopic constituents and content of glucose, bile salts, blood and glutamic-oxalacetic transaminase. The renal concentrating ability was assessed by measuring the specific gravity and volume of the urine produced during a 6-hr period of water deprivation and that produced in a 4-hr period commencing 16 hr after an oral water load of 25 ml/kg. At 2 yr the volume and specific gravity of the urine produced in the first 2 hr after the water load was also measured. Autopsies were carried out during the study on any rats killed due to ill health and on any that died, unless this was precluded by extensive autolysis or cannibalism. At 2 yr, all surviving rats were killed by exsanguination under barbiturate anaesthesia and autopsies were conducted. The animals were examined for macroscopic abnormalities and the brain, pituitary, thyroid, heart, liver, spleen, kidneys, adrenal glands and gonads were weighed. Samples of these organs together with samples of salivary gland, trachea, lung, aorta, skeletal muscle, lymph nodes, oesophagus, stomach, duodenum, ileum, caecum, colon,

LONG-TERM FEEDING STUDY ON CHOCOLATE BROWN FB

5

rectum, bile duct, bone marrow, spinal cord, pancreas and uterus and any other tissue that appeared abnormal were preserved in 10 ~o buffered formalin. These tissues were embedded in paraffin wax and sections were stained with haematoxylin and eosin for microscopic examination. Sections of kidney were also stained by the periodic acid-Schiff (PAS) technique, Schm6rl's method for lipofuscin, Perls' method for iron and the Fontana silver impregnation method. RESULTS Approximately 45 ~ of the rats of both sexes died or were killed in extremis during the study (Table 1). The majority of these deaths occurred during the last 6 months, the overall mortality being 13 ~ in males and 19 ~o in females at wk 80. At the end of the study there was no statistically significant difference between treated and control groups in the number Table 1. Mortality rates in rats f e d diets containing 0-30,000 ppm Chocolate Brown FB for 2 yr Total no. o f d e a t h s Males fed dietary levels (ppm) o f

Time (wk)

0

1000

3000

U p to 40 48 56 64 72 80 88 96 104

0 0 0 1 2 2 6 13 14

1 1 1 1 1 3 9 10 11

1 I 1 1 1 2 4 9 14

F e m a l e s fed dietary levels (ppm) o f

10,000 30,000 1 1 1 2 3 4 5 10 17

3 3 5 5 8 8 9 11 13

0

1000

3000

0 0 0 0 0 4 9 11 11

0 1 1 1 5 7 12 15 17

1 1 1 1 5 5 9 11 15

10,000 30,000 2 2 4 5 8 11 13 15 17

0 0 1 1 1 2 3 6 7

The values represent the number of rats dead or killed #~ extremis from groups of 30 animals. Numbers of deaths in groups of treated animals did not differ significantly (Chi-squared) from those in control groups.

of survivors, but during the first 18 months of feeding there was a slight, but not statistically significant, increase in the death rate of male rats fed the diet containing 30,000 ppm Chocolate Brown FB and o f females fed the diet containing 10,000 ppm compared with that of the controls. Body-weight gain was not affected by the feeding of levels o f up to 10,000 ppm but it was slightly reduced at the 30,000-ppm level (Table 2). The difference was statistically significant only in females from wk 52 to 78. There were no marked or consistent differences in food intake between treated and control animals and the mean daily intake of the colouring was approximately 40, 110, 390 and 1260 mg/kg/day in males and 50, 140, 450 and 1430 mg/kg/ day in females at the four dietary levels. Table 3 records the calculated total amounts o f colouring consumed by wk 70 and by wk 100. At wk 70 most of the rats were alive and the values given thus represent the minimum intake o f any of the animals examined, while values at 100 wk represent the total intake by surviving rats.

I. F. GAUNT, P. G. BRANTOM, P. GRASSO, MARGARET CREASEY a n d s. D. GANGOLLI Table 2. Body weight of rats fed dietary levels of O-30,O00 ppm Chocolate Brown FB for 2 yr Dietary level (ppm)

Body weight (g) at wk 0t

6

14

26

0 1000 3000 10,000 30,000

96 96 97 96 97

301 300 300 302 298

433 429 428 438 423

500 478 487 504 486

0 I000 3000 10,000 30,000

89 91 91 91 90

205 206 209 209 205

261 264 266 269 260

292 303 296 306 288

40

52

66

78

94

98

Males 555 528 539 562 530

597 570 574 608 575

628 609 613 648 611

643 615 632 665 615

610 599 615 594 585

602 592 582 587 596

Females 325 337 328 340 312

341 358 347 367 322*

377 392 387 414 353*

423 433 410 438 385*

413 429 405 476 401

425 418 400 480 393

tFirst day of feeding. Values are the means of the body weights of all surviving animals in each group. Those marked with an asterisk differ significantly (Student's t test) from those of controls: * P < 0"05.

The haematological investigations (results of which at wk 51 and 104 are given in Table 4) did not reveal any changes in the haemopoietic system attributable to the ingestion of Chocolate Brown FB. No abnormal constituents were found in the urines tested after the feeding of the colouring for 1 or 2 yr. There were no differences between treated and control rats in the concentrating ability of the kidneys (Table 5) or in the levels of the serum constituents examined (Table 6). Both the absolute and relative spleen weights (Table 7) of males fed on the diet containing 30,000 ppm Chocolate Brown FB were higher than those of the controls. There were no other changes in organ weight that could be ascribed to the treatment. A wide range of histopathological findings was common to both test and control animals (Table 8). Many of the rats showed chronic renal degeneration consisting of tubular atrophy, tubular dilatation and cast formation, together with glomerular atrophy, capsular oedema and crescent formation in some cases. Chronic myocardial fibrosis was seen in seven rats and the changes in the lungs consisted mainly of foci of peribronchial and perivascular mononuclear cell infiltration. Table 3. Total weight of colouring consumed by rats fed dietary levels of 1000-30,000 ppm

Chocolate Brown FB for 2 yr Total weight of colouring (g/rat) consumed Period of feeding (wk) 0-70 0-100

Males fed levels (ppm) of

Females fed levels (ppm) of

1000

3000

10,000

30,000

1000

3000

10,000

30,000

9 13

28 39

92 131

286 414

7 10

21 31

71 104

207 301

Values are calculated from food-intake data.

LONG-TERM FEEDING STUDY ON CHOCOLATE BROWN FB

7

T h e only histological finding that c o u l d be a t t r i b u t e d to the feeding o f C h o c o l a t e B r o w n F B was p i g m e n t d e p o s i t i o n (Table 9). This o c c u r r e d in the ceils o f the renal tubules o f m o s t rats fed o n diets c o n t a i n i n g 3000 p p m a n d above. T h e p i g m e n t was b r o w n in co l o u r , c o a r s e l y g r a n u l a r and fairly evenly distributed t h r o u g h o u t t h e cytoplasm. It gave negative Table 4. Haematological values of rats fed dietary levels of 0-30,000 ppm Chocolate Brown FB for 1 or 2 yr

Leucocytes Dietary level (ppm)

No. of rats examined

Hb (g/100 ml)

MetHb ( ~ of Hb)

PCV (~)

RBC (106/ mm 3)

Retics ( ~ of RBC)

Total (10a/ mm a)

Differential (~.) N

E B

2 --2 5

L M

Wk 51 Male 0 1000 3000 10,000 30,000

12 12 12 12 12

16.0 15.8 15.4 15.9 13.2

49 48 48 51 48

8.05 7.78 8.30 8.33 7-47

0.7 --1.0 2.4

14-4 13.5 13.6 12.0 12.5

17 --17 20

Female 0 1000 3000 10,000 30,000

12 12 12 12 12

15"1 14-2 14"2 14"1 13-3

44 45 44 47 44

7.30 7'27 7.23 7.07 6"53

1.1 --0.9 1.1

10"1 12.1 10'2 10"4 10.2

16 4 0 76 4 0 -- -- 0 16 3 0 74 7 23 5 0 65 7

Male 0 1000 3000 10,000 30,000

16 19 14 13 16

12.9 14.1" 13.9 14.6"* 13.3

4-2 3.6 3.7 3"6 3.1

Wk 104 41 7.36 43 7.85 43 7.57 44* 8-26* 42 7.84

2.0 1.4 1.1 0.9 1.7

8-2 6.8 6.8 6.0 5.9

38 2 0 55 42 2 0 52 36 1 1 55 33 3 I 57 42 4 1 48

5 4 7 6 5

Female 0 1000 3000 I0,000 30,000

19 14 18 15 24

12"4 12-2 11"9 11"9 12"3

3"7 4"5 4-2 4"0 4"0

39 38 40 40 39

1"4 2.4 1-8 2.5 1.9

4'4 3'9 3"6 4"2 3"9

38 35 33 41 36

5 4 6 6 5

6-90 7"03 6"86 6.31" 6"55

2 2 3 2 8

0 74 0 0 0 73 0 70

0 0 0 0 1

55 59 58 51 50

7 8 5

Hb = Haemoglobin MetHb = Methaemoglobin PCV = Packed cell volume RBC = Red blood cells Retics= Reticulocytes N = Neutrophils E = Eosinophils B = Basophils L ----Lymphocytes M = Monocytes. Values are means of the numbers of rats shown. Those marked with asterisks differ significantly (Student's t test) from those of controls: *P<0-05; **P<0"01. No inclusions were seen in the erythrocytes.

r e a c t i o n s o n staining by th e P A S , Schm6rl, Perls a n d F o n t a n a m e t h o d s . A t the highest level (30,000 p p m ) there was also p i g m e n t d e p o s i t i o n in the l y m p h nodes, p a r t i c u l a r l y in females. I n a few female rats p i g m e n t was also f o u n d in the cells o f the intestinal m u c o s a a n d spleen an d in the K u p f f e r cells o f the liver. T h e t u m o u r s e n c o u n t e r e d (Table 10) were m a i n l y benign m a m m a r y f i b r o a d e n o m a s . A d e n o m a s o f the pituitary, o f the Islet cells o f the pancreas, a n d o f the lung, t h y r o i d a n d

8

I.F. GAUNT, P. G. BRANTOM, P. GRASSO, MARGARETCREASEYa n d s. D. GANGOLLI

adrenals were also e n c o u n t e r e d . M a l i g n a n t t u m o u r s were very rare, consisting o f o n e retroperitoneal fibrosarcoma, f o u r l y m p h o s a r c o m a s (two i n the intestine, o n e i n the t h y m u s a n d one generaliTed) a n d one c a r c i n o m a o f the uterus. T h e t u m o u r type a n d incidence could n o t b e related to t r e a t m e n t with Chocolate B r o w n FB. Table 5. Results o f urine analysis of rats fed Chocolate Brown FB at 0-30,000 ppm in the diet for I and 2 yr Concentration test Sex and dietary level (ppm)

No. of rats examined

Specific gravity GOT (IU)

0-6 hr

16-20 hr

Dilution test (2 hr)

Volume (ml) 0-6 hr

16-20 hr

Specific gravity

Volume (ml)

---

Wk 52 Male 0 1000 3000 10,000 30,000

30 29 29 29 25

5-3 8.6 8.4 6.1 6.7

1.055 1-059 1.057 1.044 1.048

1.087 1.091 1.084 1.084 1-082

4.4 4.2 3-4 5-2 4.0

1.3 1-2 1.8 1-I 1.2

---m --

Female 0 1000 3000 10,000 30,000

30 29 29 27 30

7"1 8.4 7-1 6.9 7.5

1.065 1.062 1.056 1-047 1.057

1.100 1.087 1-087 1.073 1.076

2.5 3.8 3.9 4.4" 2.7

0.3 1-1 0.9 1.2 1-0

-m --m

Male 0 1000 3000 10,000 30000

12 12 12 12 12

5.3 6-7 7.7 7.7 8.9

Wk 102-104 1.023 1.040 1.027 1-045 1.023 1.049 1.025 1.042 1.027 1.057

6-6 6.4 3.9 4.8 5.1

2.7 3-4 2.5 3.2 2-4

1.010 1.010 1.013 1.009 1.015

5.4 5-1 4.5 4.6 5.1

Female 0 1000 3000 I0,000 30,000

12 12 12 12 12

5.3 6.4 -7.8 8-5

1.026 1.025 1.035 1.021 1"022

4.8 3.8 2.9 4.7 4"6

2.5 1.1 1.4 2.1 1"7

1.010 1.005 1-005 1.006 1.009

5.6 5.8 5.1 6.6 5"5

1.058 1.059 1-055 1.038 1"053

--

__ --

GOT ----Glutamic-oxalacetic transaminase Values are means for the numbers of rats shown. Those for treated animals do not differ significantly (Student's t test) from those of controls. Tests for blood, bile salts and reducing substances were negative in all groups.

DISCUSSION T h e results presented show t h a t the death rate a t the e n d o f the 2-yr feeding p e r i o d was n o t affected by the i n c l u s i o n o f Chocolate B r o w n F B i n the diet o f rats at levels o f u p to 30,000 p p m . A t the highest dietary level the deaths i n males occurred slightly earlier t h a n i n other g r o u p s ; this was n o t f o u n d i n the females, which i n the 30,000-ppm g r o u p e x h i b i t e d the lowest d e a t h rate t h r o u g h o u t the study. The earlier d e a t h o f the males was n o t d u e to t h e toxicity o f Chocolate B r o w n FB. F o u r males o f the 30,000-ppm g r o u p died before w k

LONG-TERM FEEDING STUDY ON CHOCOLATE BROWN FB Table 6. Results of serum analysis of rats fed dietary levels of O-30,O00 ppm

Chocolate Brown FB for 2 yr Dietary level (ppm)

No. of rats examined

0 1000 3000 10,000 30,000

16 19 14 13 16

0 1000 3000 10,000 30,000

19 14 18 15 24

Urea (mg/100 ml)

GOT (IU)

GPT (IU)

Males 19"9 18"9 19.5 20"3 18"9

53'5 50"2 47.7 55"6 53'1

17.7 14"6 14"0 14.2 15"4

Females 20-8 20"0 18"4 20"9 21-5

55"4 51"0 53-1 52"3 52"4

18"4 13"9 17-7 15"0 15"0

GOT = Glutamic-oxalacetic transaminase GPT = Glutamic-pyruvic transaminase Values are means for the numbers of rats shown. Those for treated animals do not differ significantly (Student's t test) from those of controls.

80 from a pulmonary infection, which had probably been introduced into the cage in which all four were housed. In addition, two male animals dying in the early stages showed haemorrhages, one in the testes and the other in the intestinal tract and thorax. A few months after these deaths, there was an outbreak of a haemorrhagic condition among male rats o f the colony associated with dietary vitamin-K deficiency (Gaunt & LanePetter, 1967). It is probable that, in the early stages o f the experiment, all the animals in this study were suffering from some degree of vitamin K deficiency. In their study, Gaunt & Lane-Petter (1967) found that haemorrhages occurred first in rats fed the highest dose of a test compound irrespective of its toxicity and later in rats fed lower dosage levels and in controls. It is possible, therefore, that the high dietary concentration of Chocolate Brown FB was sufficient to precipitate haemorrhage in these two cases. Females fed the 10,000-ppm Chocolate Brown FB diet showed an increased mortality during wk 68-76. However, these figures include animals that were killed because they bore large mammary fibroadenomas but that were otherwise in good health. If these rats are excluded from the analysis the mortality figures at wk 76 are: control, 0; 1000 ppm, 1; 3000 ppm, 1 ; 10,000 ppm, 3; 30,000 ppm, 0. Mammary fibroadenomas occur frequently in this strain o f rat (Grasso, Lansdown, Kiss, Gaunt & Gangolli, 1969). The higher incidence at the I0,000 ppm level cannot be attributed to Chocolate Brown FB treatment since after the 2-yr treatment period the total incidence of this tumour was not increased in treated animals and the incidence in females fed at the highest dietary level was among the lowest found in any group. It is concluded that the inclusion of Chocolate Brown FB in the diets o f rats at levels of up to 30,000 ppm does not lead to an increase in the death rate in rats. There was a slight retardation of body-weight gain without any marked reduction in food intake at the 30,000 ppm level. A similar observation was made in a short-term feeding test (Gaunt et al. 1967), although in the present study a reduction in weight gain was not seen until after treatment for longer than 13 wk.

16 19 14 13 16

19 14 18 15 24

16 19 14 13 16

19 14 18 15 24

Male 0 1000 3000 10,000 30,000

Female 0 1000 3000 1~000 30,000

Male 0 1000 3000 10,000 30,000

Female 0 1000 3000 10,000 30,000

0.46 0.47 0"54 0.41 0.51

0-38 0.38 0.40 0.37 0.38

1.78 1.81 1"83 1-82 1.87"

1-96 2.04 1.89 1.97 2.01

Brain

0-31 0.34 0"36 0"30 0"36

0.36 0.34 0.39 0.36 0.38

1.28 1"33 1"30 1"33 1"25

1.84 1.84 1.83 1.92 2.01"

Heart

Kidneys

0-74 0"72 0"63 0"85 0"69

1"01 1"13 1"01 1"17 1"25" 2.65 2"66 2"61 2"76 2"51

5"42 4"59 5"32 4"90 4"84

Absolute organ weights (g)

Spleen

78 76 77 82 74

86 81 85 80 73

Adrenalst

3"24 3.12 2.95 3"25 3.08

3.27 2-89 3.41 2"87 2"95 0"19 0.18 0.20 0"19 0"21

0.20 0-21 0.21 0"22 0"25* 0"70 0"68 0.76 0"65 0-70

1.07 0.84* 1.18 0"94 0"92

26 20 24 18 26

17 18 18 16 16

Relative organ weights (g/100 g body we|gh0

12.84 12.58 I 1"05 14.75 11"15

16"64 16"63 16"04 15"32 15"50

Liver

Organ weight

49 44 58 37 52

0-75 0.74 0.65 0"65 0"85

150 170 190 180 190

3"87 3"96 3"21 3"48 4"58

Gonads+*

4"9 4"9 6"5 3"4 3"9

2.6 2.8 2.7 2"8 2"8

15 18 21 16 14

14 15 14 15 15

Pituitaryt

7.6 8'0 8.1 6.4 7.4

7.5 7.3 7.6 8"5 8"5

29 30 28 27 27

47 40 38 47 45

Thyroid'l"

396 411 364 455 362

520 538 481" 535 531

Terminal body weight (g)

"['Weights of this organ are expressed in mg and rag/100 g body weight. ++Weights of female gonads are expressed in mg and mg/lO0 g body weight. Values are means of the numbers of animals shown and those marked with an asterisk differ significantly (Student's t test) from those of controls: *P < 0"05

No. of rats examined

Dietary level (ppm)

Table 7. Organ weights of rats fed dietary levels o./'0-30,000 ppm Chocolate Brown FB for 2 yr

0

> Z

U

m ,<

t'rl

¢'1

m

9

~0 >

P~

Z ,..] 0

.m

>

No. of rats examined...

Skin Chronic inflammatory cell infiltration Heart Fibrosis with foci of chronic inflammatory cell infiltration Pancreas Chronic inflammatory cell infiltration Lung Chronic inflammatory cell infiltration Liver Fatty degeneration Necrosis Thyroid Nodular hyperplasia Kidney Nephropathy Prostate Chronic inflammatory cell infiltration Uterus Chronic inflanwnatory cell infiltration of the endometrium

Tissue and type of abnormality

1 1

0 9 0 0 0 28 0

1

0 10 0 0 2 24 0

1 0 12 1 0 1 27 0

28

3000

0

28

I000

0

29

0

1

24

0

2 0

12

0

0

0

29

I0,000

Males fed dietary levels (ppm) of

0

23

0

0 I

10

0

0

0

30

30,000

0

8 0 1 1

5

I

5 2 1 1

25

0

1

1

0

o

29

1000

o

29

0

0

6

2

0 1

10

0

0

o

30

3000

2

4

0

3 0

7

0

1

o

29

10,00

Females fed dietary levels (ppm) of

No. of animals affected

Table 8. Incidence of histological findings (excluding tumours) in rats fed diets containing 0-30,000 ppm Chocolate Brown FB for up to 2 yr

0

12

0

1 2

4

0

1

o

29

30,000

O o

,~ o Z

~.

Z

m

9- I

t", 0 Z

Kidney Liver Spleen Lymph nodes Intestine

Organ

No. ofrats examined... 0 0 0 0 0

29

0

0 0 0 0 0

28

1000

27 0 0 0 0

28

3000

25 0 0 0 0

29

10,000

Males fed dietary levels (ppm) of

29 0 0 2 0

30

30,000

0 0 0 0 0

29

0

0 0 0 0 0

29

1000

29 0 0 0 0

30

3000

27 0 0 0 0

29

10,000

Females fed dietary levels (ppm) of

No. of rats with pigmented organs

Table 9. bzcidence of pigmentation in organs of rats fed dietary levels of O-30,O00 ppm Chocolate Brown FB for 2 yr

29 2 1 12 2

29

30,000

o

>

t~

m

! >

O

O ~

> Z

No. of rats examined...

Skin Fibroma Squamous cell papilloma Pancreas Islet cell adenoma Lung Adenoma Thyroid Adenoma Stomach Fibroma Adrenal gland Adenoma Ovary Benign thecoma Pituitary Adenoma Mammary gland Fibroadenoma Vagina Fibroma Uterus Fibromyoma Carcinoma Lympho-reticular system Lymphosarcoma Retroperitoneal tissue Fibrosarcoma

Organ and type of turnout

0 0 3 0 0

0 4

0 0

0 0

3 0 1

0 1

2 0

2 0

28

1000

0 0

29

0

0

0

0

0

0

1

2

0

1

0 0

28

3000

0

1

0

2

1

0

0

0

1

0 0

29

10,000

Males fed dietary levels (ppm) of

0

0

0

0

0

0

0

0

0

0 0

30

30,000

2

0

0 0

0 0

1

0

0

1

0

1

15

20

0

0

0

1

0

0

0 0

0

0

0 1

29

1000

0

0

0 0

29

0

0

0

0 0

0

20

0

0

0

0

0

0

1

0 0

30

3000

0

0

0 1

0

21

0

0

0

0

0

2

0

! 0

29

10,000

Females fed dietary levels (ppm) of

No. of animals with turnouts

Table 10. Incidence of tumours in rats fed dietary levels of 0-30,000 ppm Chocolate Brown FB for 2 yr

0

0

1 0

0

17

0

0

I

0

!

1

0

0 0

29

30,000

0

m

= O o >

O Z

Z

m

m

9

O Z

14

I . F . GAUNT, P. G. BRANTOM, P. GRASSO, MARGARET CREASEY and s. D. GANGOLLI

The only histological finding which could be attributed to the treatment with Chocolate Brown FB was the presence o f pigment in various tissues. This occurred at all dosage levels from 3000 ppm upwards. In the short-term toxicity test (Gaunt et al. 1967) the pigment was present only at dietary levels of I0,000 and 30,000 ppm. Because of the negative staining reactions the pigment is thought to be a protein complex of either the colouring itself or its metabolic products rather than lipofuscin. Miller & Palade (1964) showed that cells of the proximal renal tubule were capable o f absorbing protein from the glomerular filtrate into lysosome-like bodies. Chocolate Brown FB itself binds with serum proteins and it is possible that it may be taken up by lysosomes together with the protein. However, Gangolli (1969) showed that the protein-binding ability of Chocolate Brown FB was the lowest (0.01 g colouring/100 g protein) of a series of azo colourings used in food. It would seem possible, therefore, that protein-bound metabolic products may have contributed to the pigment seen. The presence of this pigment did not appear to cause injury to the kidneys since it was not associated with changes in the weight, histological appearance or function of the kidneys even after the colouring had been ingested for the major part o f the lifespan. The other lesions, including tumours, were randomly distributed between test and control animals and could not be attributed to treatment with Chocolate Brown FB. This test produced no evidence that Chocolate Brown FB has any carcinogenic potential in rats even when fed at a level of 30,000 ppm in the diet. The no-untoward-effect level in the diet o f rats over a 2-yr period was 1000 ppm (approximately 50 mg/kg/day). The authors wish to thank Mrs. G. Wexler for assistance with the statistical analyses in this study. They are also grateful to the staff of the BIBRA Animal House for maintenance of the experimental animals and to the staff of the Analytical Chemistry, Pathology and Toxicology Departments for technical assistance. Acknowledgements

REFERENCES Food Standards Committee (1964). Report on Colouring Matters. HMSO, London. Fore, H., Walker, R. & Golberg, L. (1967). Studies on Brown FK. II. Degradative changes undergone in vitro and in vivo. Fd Cosmet. Toxicol. 5, 459. Gangolli, S. D. (1969). Studies on the action of certain surface active substances on the cell membrane. Ph.D. Thesis, London University. Gaunt, I. F., Hall, D. E., Farmer, Madge & Fairweather, F. A. (1967). Acute (mouse and rat) and shortterm (rat) toxicity studies on Chocolate Brown FB. Fd Cosmet. Toxicol. 5, 159. Gaunt, I. F. & Lane-Petter, W. (1967). Vitamin K deficiency in 'SPF' rats. Lab. Anim. 1, 147. Grasso, P., Lansdown, A. B. G., Kiss, Ida S., Gaunt, I. F. & Gangolli, S. D. (1969). Nodular hyperplasia in the rat liver following prolonged feeding of Ponceau MX. Fd Cosmet. Toxicol. 7, 425. Joint FAO/WHO Expert Committee on Food Additives--Eighth Report (1965). Specifications for the Identity and Purity of Food Additives and their Toxicological Evaluation: Food Colours and some Antimicrobials and Antioxidants. Tech. Rep. Ser. Wld Hlth Org. 309. Miller, F. & Palade, G. E. (1964). Lytic activities in renal protein absorption droplets. J. cell Biol. 23, 519. Sunderman, F. W. & Sunderman, F. W. Jr. (1960). Hemoglobin, Its Precursors and Metabolites. p. 53, Lippincott, Philadelphia, U.S.A.

LONG-TERM FEEDING STUDY ON CHOCOLATE BROWN FB

Etude nutritionnelle ~ long terme, sur des rats, du Brun Chocolat FB R~um~----Deux groupes de jeunes rats sevrgs, 30 mfiles et 30 femelles, ont re~u pendant deux ans du Brun Chocolat FB b. raison de 0 (animaux t6moins), I000, 3000, 10 000 ou 30 000 ppm du r6gime. Aucun effet imputable au traitement n ' a 6t6 observ6 sous le rapport de la mortalit6, du gain de poids, de l'hgmatologie, de la chimie du s&um, du poids des organes et de la fr6quence de tumeurs. Des dgp6ts de pigments ont 6t~. d~cel~s dans les cellules tubulaires du rein chez les animaux soumis aux r6gimes h 3000 ppm et plus. Chez quelques femelles soumises au r6gime b. 30 000 ppm on a aussi retrouv6 le pigment dans les cellules de Kuppfer du foie et dans les cellules des nodules lymphatiques, de la rate et de la muqueuse intestinale. Le Brun Chocolat FB n'a manifest6 aucun pouvoir canc6rig6ne et son seuil d'indiff&ence gtait de 1000 ppm (environ 50 mg/kg/jour).

Langzeitiger Verfiitterungsversuch mit Chocolate Brown FB an Ratten Zusammenfassung---Gruppen von 30 m~innlichen und 30 weiblichen abgesetzten Ratten erhielten 2 Jahre lang Futter mit 0 (Kontrolle), 1000, 3000, 10 000 und 30 000 ppm Chocolate Brown FB. Es wurden keine dieser Verabreichung zuzuschreibenden Erscheinungen bei der Mortalitht, K6rpergewichtszunahme, H/imatologie, Serumchemie, den Organgewichten und der Tumorh~iufigkeit gefunden. In den Tubuluszellen der Niere wurde bei Konzentrationen yon 3000 ppm und h6her eine Pigmentabscheidung festgestellt. Pigment wurde auch in den Kupfferschen Zellen der Leber und in Zellen der Lympb_knoten, der Milz und der Darmschleimhfi.ute einiger weiblicher Ratten gefunden, an die Futter mit 30 000 ppm verftittert worden war. Eine carcinogene Wirkung wurde bei Chocolate Brown FB nicht festgestellt. Die yon schhdlichen Wirkungen freie Konzentration war 1000 ppm (etwa 50 mg/kg/Tag).

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