Long-term feeding study on black PN in rats

Fd Cosmet. ToxicoL Vol. 10, pp. 17-27. Pergamon Press 1972. Printed in Great Britain

Long-term Feeding Study on Black

PN

in

Rats

I. F. GAUNT and F. M. B. CARPAlS--Toxicology P. GRASSOand IDA S. KIss Pathology and

S. D. GANGOLLI--Analytical Chemistry British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, England (Received 5 July 1971) Abstract--Groups of 24 male and 24 female weanling rats were fed for 2 yr on diets containing 0 (control), 1000, 5000 or 10,000 ppm Black PN. No effects attributable to treatment were found in respect of mortality, body-weight gain, food intake, haematology, serum chemistry, renal concentration tests, organ weights or incidence of pathological findings, including tumours. No carcinogenic potential was detected in Black PN and the no-untoward-effect level was I0,000 ppm (approximately 500 mg/kg/day).

INTRODUCTION Black PN (Brilliant Black BN; C.I. (1956) no. 28440) is a food colouring consisting principally of the tetrasodium salt of 8-acetamido-2-(7-sulpho-4-p-sulphophenylazo-lnaphthylazo)-l-naphthol-3,5-disulphonic acid (Fig. 1). It is permitted for use in the UK (The Colouring Matter in Food Regulations 1966, Statutory Instrument 1966, no. 1203), but the reports of the Food Standards Committee (1964) and the Joint FAO/WHO Expert Committee on Food Additives (1965) expressed a need for further evidence of its safety-inuse.

OH NHCOCHs

S03No FIG. 1. Chemicalstructure of the principal constituentof Black PN. Black PN is readily broken down to colourless compounds when incubated in vitro for 19 hr with rat caecal contents (Walker, 1968). Ryan & Welling (1970) demonstrated that, on oral administration to rats, both azo bonds readily undergo reductive cleavage in the caecum, with formation of 8-acetamido-l-hydroxy-2-naphthylamine-3,5-disulphonic acid (I) and sulphanilic acid (II), both thought to be excreted unchanged, and 1,4-diaminonaphthalene-6-sulphonic acid (III) and its N-acetylated conjugates. All these metabolites were detected in the faeces, but only II and the N-acetylated conjugates of III were detected in the urine. 17

18

I . F . GAUNT, F. M. B. CARPANINI, P. GRASSO, IDA S. KISS and s. D. GANGOLLI

When Black PN was administered intravenously, 1-(4-sulphophenylazo)-4-naphthylamine-6-sulphonic acid (IV) with some N-acetylated conjugates of III were detected in the bile (Ryan & Welling, 1970), suggesting that metabolism occurred in the liver. The biliary metabolites were thought to be further metabolized by the intestinal flora of the rat. These authors also demonstrated the presence of sulphanilic acid in the urine of a man dosed with Black PN, showing that azo reduction can occur in man. The acute oral LDso is greater than 5 g/kg in mice (Deutsche Forschungsgemeinschaft-Farbstoff-Kommission, 1957) and rats (Gaunt, Farmer, Grasso & Gangolli, 1967). The latter authors found that the intraperitoneal LDso was in the range of 0-5-1.0 g/kg in mice and 0.9-1.2 g/kg in rats and that after oral administration black material was excreted in the faeces, suggesting that Black PN was not absorbed or was not completely metabolized when large doses were given. Gaunt et al. (1967) fed Black PN to rats at dietary levels of 3000-30,000 ppm for 90 days and established a no-untoward-effect level of 10,000 ppm. Male animals fed on diets containing 30,000 ppm Black PN showed growth retardation, reduced food intake and increases in relative kidney and testis weights. Gaunt, Colley, Creasey & Grasso (1969) fed Black PN to pigs at dietary levels providing an intake of 100-900 mg/kg/day for 90 days and found a no-untoward-effect level of 100 mg/kg/day. Cysts containing mucus and fibrin were found in the mucosa of the ileum of four of the six pigs given 900 mg/kg/day and one of the six given 300 mg/kg/day. These were attributed to the irritant nature of high concentrations of the material. No carcinogenic effects were found in long-term studies reported by the Deutsche Forschungsgemeinschaft--Farbstoff-Kommission(1957). In these, rats were fed on a diet containing 1000 ppm Black PN for 410 days with subsequent observation to 761 days or were maintained on drinking water containing 5000 ppm Black PN for 502 days with observation to 923 days. One of ten rats developed a fibromyoma following twice-weekly subcutaneous injection of 0.5 ml of a 10~o solution of Black PN for 1 yr. This paper presents the results of a long-term feeding study in rats carried out as part of the BIBRA safety evaluation programme. EXPERIMENTAL Materials. Black PN was supplied through the Chemical Industries Association and complied with the following specification of the British Standards Institution (1968):

Composition, essentially tetrasodium 8-acetamido-2-(7-sulpho-4-p-sulphophenylazo1-naphthylazo)-l-naphthol-3,5-disulphonate; dye content*, rain 82~o; subsidiary dyes*, max 4 Yo; matter volatile at 135°C*, max 10 ~o; matter insoluble in water*, max 0-1~o; matter soluble in diisopropyl ether*, max 0"2Yo; chloride and sulphate (as sodium salts)*, max 8"0~o; copper*, max 10 ppm; arsenic*, max 1 ppm; lead*, max 10 ppm; heavy metals (as sulphides)*, producing a colour no more intense than that of the reference standard. Animals and diet. Rats of the CFE strain obtained from an SPF colony were maintained on Spillers' Laboratory Small Animal Diet and water ad lib. The animal rooms were maintained at 21 4- I°C with a relative humidity of 50--60~. *By methods of analysis as described in BS 3210:1960 (Methods for the Analysis of Water-soluble Coal-tar

Dyes Permittedfor Use in Foods).

LONG-TERM FEEDING STUDY ON BLACK PN IN RATS

19

Experimental design and conduct. Groups of 24 male (body weight 115-140 g) and 24 female (body weight 115-130 g) rats housed four to a cage were fed on diets containing 0 (control), 1000, 5000 or 10,000 ppm Black PN for up to 2 yr. The general condition and behaviour of the animals were observed and any animal that was in ill health was isolated, to be returned to its cage on recovery or to be killed when moribund. The rats were weighed and the food intake was recorded every 2 wk up to wk 101. At wk 18, 35 and 82, samples of blood were collected from the lateral caudal veins of 12 rats of each sex from each dietary level. At wk 54 blood was collected from the same number of rats from the controls and those fed on diets containing 5000 or 10,000 ppm Black PN, and at 2 yr from the aorta of all surviving rats. All blood samples were examined for haemoglobin concentration, packed cell volume and counts of erythrocytes and total leucocytes. Counts of reticulocytes, erythrocytes with Heinz bodies and the various types of leucocyte were carried out on all blood samples collected at 2 yr but only on samples from the controls and the two top dietary levels of Black PN (5000 and 10,000 ppm) at the earlier examinations. In addition, the methaemoglobin concentration (Sunderman & Sunderman, 1960) was determined in samples collected at 2 yr. The renal concentrating ability was assessed in six males and six females from each dietary level at 12 months and in all survivors at 2 yr. Measurements were made of the specific gravity and volume of urine produced in a 6-hr period of water deprivation and of that produced between 16 and 20 hr after a water load of 25 ml/kg. The urinary glutamicoxalacetic transaminase concentration was determined in the samples obtained at 2 yr. After 2 yr, the surviving rats were killed by exsanguination under barbiturate anaesthesia. An autopsy was conducted during which macroscopic abnormalities were noted and the brain, pituitary, thyroid, heart, liver, spleen, kidneys, adrenals and gonads were weighed. Samples of these organs and of salivary glands, thymus, lymph nodes, pancreas, urinary bladder, lungs, stomach, small intestine, colon, caecum, muscle, uterus and any other tissue which appeared abnormal were preserved in 10 Yo buffer formalin. Paraffin wax sections of these tissues were stained with haematoxylin and eosin for microscopic examination. Autopsies were conducted on all the rats that died or were killed because of ill health during the experiment. Samples of all the above organs were taken and examined for microscopic changes except when this was prevented by advanced autolysis. Serum collected at autopsy was analysed for urea (Marsh, Fingerhut & Miller, 1965) and for glutamic-oxalacetic and glutamic-pyruvic transaminases (Karmen, 1955). RESULTS The fur and faeces of the rats fed diets containing Black PN were coloured black. There was no black colour in urine collected free of faecal or other contamination. Many rats died or were killed during the course of the study but at no time were the numbers of deaths in the Black PN-treated animals significantly greater than those of the controls. The majority of the deaths occurred in the last 6 months of treatment (Table 1). There were no significant differences between treated and control rats in body-weight gain (Table 2) or food consumption. The mean daily intakes of Black PN were approximately 40, 180 and 360 mg/kg/day in males and 45, 240 and 470 mg/kg/day in females at the three dietary levels. The calculated total amounts of the colouring consumed by wk 69 and 102 are shown in Table 3. The figures quoted for wk 69 represent the minimum intake by any of the animals examined, since most of the animals were still alive at this time. The figures at wk 104 represent the maximum amount consumed.

20

I . F . GAUNT, F. M. B. CARPANINI, P. GRASSO, IDA S. KISS a n d s. D. GANGOLLI Table 1. Cumulative death rate in rats fed dietary levels of O-lO,O00 ppm Black P N for up to 2 yr Total no. of deaths Males fed levels (ppm) of

Duration of feeding

Females fed levels (ppm) of

(wk)

0

1000

5000

I0,000

0

1000

5000

10,000

10 20 30 40 48 56 64 72 80 88 96 104

0 0 0 1 2 3 3 3 4 9 16 19

0 0 0 0 0 0 0 I 6 7 10 14

0 1 2 2 2 3 7 7 8 9 14 18

1 1 1 1 2 2 2 2 5 7 I0 13

0 0 0 0 0 0 0 0 1 2 5 10

0 0 0 0 1 1 1 2 2 3 4 7

0 1 1 1 1 2 2 3 3 4 8 14

0 0 0 0 0 ! I 1 2 7 I0 16

Numbers represent the total number of rats dead or killed in extremis from groups of 24 animals. Numbers of deaths in groups of treated animals did not differ significantly (Chi-squared) from those in control groups.

R e s u l t s o f t h e h a e m a t o l o g i c a l studies c a r r i e d o u t at w k 54, 82 a n d 104'are g i v e n in T a b l e 4. N o a d v e r s e h a e m a t o l o g i c a l c h a n g e s w e r e seen in e i t h e r sex o r at a n y d i e t a r y level u p to 12 m o n t h s . A t w k 82, t h e h a e m o g l o b i n c o n c e n t r a t i o n a n d p a c k e d cell v o l u m e w e r e r e d u c e d in f e m a l e s fed a diet c o n t a i n i n g 10,000 p p m B l a c k P N . T h e s e effects w e r e n o t f o u n d a f t e r 2 yr, a l t h o u g h at this t i m e this g r o u p s h o w e d a r e d u c t i o n in t h e t o t a l l e u c o c y t e c o u n t . T h e t e r m i n a l h a e m o g l o b i n c o n c e n t r a t i o n a n d p a c k e d cell v o l u m e o f m a l e rats w e r e r e d u c e d at all d i e t a r y levels. T h e r e w e r e n o significant differences b e t w e e n t r e a t e d a n d c o n t r o l rats in t h e

Table 2. Mean body weight of rats fed dietary levels of O-lO,O00 ppm Black P N for 2 yr Dietary level (ppm)

Body weight (g) at wk 0*

13

25

39

51

65

77

91

101

0 1000 5000 10,000

130 131 133 139

408 412 404 403

492 496 482 496

Males 566 556 543 557

618 616 590 607

659 663 630 645

670 659 646 650

661 640 621 648

662 590 580 590

0 1000 5000 I0,000

122 121 122 123

252 254 253 256

286 288 285 289

Females 328 324 319 322

350 357 344 350

399 394 387 395

417 416 400 409

461 435 431 431

400 452 469 458

*Day 1 of feeding. Values are the means for groups of 24 animals or survivors. Statistical analyses revealed no significant differences between control and experimental animals.

LONG-TERM FEEDING STUDY ON BLACK PN IN RATS

21

Table 3. Total weight of colouring consumed by rats fed dietary levels of lO00-10,O00 ppm Black PN for 2 yr Total weight of colouring (g/rat) consumed Duration of feeding (wk)

Males fed levels (ppm) of

Females fed levels (ppm) of

I000

5000

10,000

1000

5000

0-69 0-104

I0 15

48 71

99 146

8 12

39 59

I0,000 77 115

Values are calculated from food intake data.

results of biochemical analyses of serum and urine (Table 5) or of the renal concentration test (Table 6). There were no statistically significant differences between the absolute organ weights o f control rats and those o f rats fed diets containing Black PN (Table 7). However, in male rats, the relative liver weights were significantly higher in all treated groups than in the controls (Table 7). The incidence of histological findings, excluding tumours, is shown in Table 8. Chronic rat nephropathy was found in many of the animals. This consisted of renal tubular dilation, protein casts and foci of round-cell infiltration. Chronic lung changes encountered consisted o f peribronchial and perivascular lymphocyte cuffing. The incidence o f these findings did not vary significantly between treated and control groups. The most common tumours encountered (Table 9) were adenomas of the mammary glands, but the incidence was not related to treatment. In a few rats, benign tumours were found affecting the pancreas, thyroid, ovary, adrenal or subcutaneous tissue. The only malignant tumours seen were one carcinoma of the thyroid with lung metastases in a male rat fed the diet containing I0,000 ppm Black PN and a mammary gland carcinoma in a female from the 5000-ppm group. DISCUSSION It has been shown that orally administered Black PN is metabolized in the caecum o f the rat (Ryan & Welling, 1970). The presence of coloured faeces in this study showed that, at the dosage levels used, some o f the Black P N passed through the gastro-intestinal system unchanged. Similar observations were made by Gaunt et al. (1967) in rats and Gaunt et al. (1969) in pigs. The mortality rate was high in this test (49 ~ in females and 67 ~o in males over the 2-yr feeding period), with most of the deaths occurring after 80 wk. The presence o f Black P N did not influence the number of deaths or the average lifespan and the high death rate is attributed to the nephropathy and pulmonary inflammation that were found at autopsy. The haemoglobin concentration and packed cell volume, which were slightly low at 18 months in females fed a diet containing 10,000 ppm Black PN, were normal at 2 yr, suggesting that the findings were o f no significance. Lowering o f the haemoglobin concentration and packed cell volume was found in all male experimental groups after 2 yr of feeding but the degree of depression was not related to the dosage level. The average haemoglobin concentration in male controls was higher at 2 yr than during earlier periods. Females showed decreases in this value and in the packed cell volume with increasing age, a general

22

I . F . GAUNT, F. M. B. CARPANINI, P. GRASSO, IDA S. KISS a n d s. D. GANGOLLI

p a t t e r n s e e n in b o t h s e x e s o f c o n t r o l a n d e x p e r i m e n t a l r a t s in o t h e r c o m p a r a b l e s t u d i e s w i t h f o o d c o l o u r i n g s ( G a u n t , B r a n t o m , G r a s s o , C r e a s e y & G a n g o l l i , 1972; G r a s s o , L a n s d o w n , K i s s , G a u n t & G a n g o l l i , 1969). T h e h i g h f i g u r e s f o r t h e c o n t r o l m a l e s i n t h i s s t u d y w e r e p r o b a b l y d u e t o t h e f a c t t h a t t h e y w e r e b a s e d o n e x a m i n a t i o n o f o n l y five a n i m a l s . T h e Table 4. Haematological findings in rats fed dietary levels of 0-10,000 ppm Black P N for up to 105 wk Sex and dietary level (ppm)

Leucocytes No. of rats examined

Hb (g/100 ml)

MetHb (Yo of Hb)

PCV (~)

RBC (106/ mm a)

Retics (Yo of RBC)

Total (103/ mm 3)

Differential ( ~ ) N

E

L

M

54

Male 0 1000 5000 10,000

12 12 12 12

15.2 -14"9 14"9

Female 0 1000 5000 10,000

12 12 12 12

14-0 . 14.6 14-7

-----.

49

7.73

1.74

15.0

23

2

68

7

50 50

7.81 7.90

1.09 1-22

14.9 15.0

23 21

2 4

69 72

6 3

1.28

11-0

20

2

72

6

1.25 0-95

12.6 11-0

22 13

3 2

73 81

2 4

25

3

64

8

30 27

3 3

59 61

8 9

45 .

---

. 46 48

7.11 . 7.33 6-85

.

W k 82

Male 0 1000 5000 10,000

12 12 12 12

15"6 16"0 15"5 15"8

-----

50 51 50 50

8"63 8"69 8"22 7"89

1"69 -1"31 1"41

15"5 16"9 18.2 15-2

Female 0 1000 5000 10,000

12 12 12 12

14-4 13-6 15-0 13.4"**

-----

47 47 49 45*

6.82 6.69 7-89** 7.18

1.51 -1.28 1.69

10.0 9.1 11-8 9.8

25

5

62

8

31

3

57

9

Wk 105 Male 0 1000 5000 10,000

5 9 6 11

16.2 14.2" 12.5" 14.4"*

4.93 5.90 7-78 5.35

48 43* 38* 45

7.36 6.79 6.12 6"96

0.94 1.31 0.93 0.77

8.5 7.2 8.5 7.6

39 46 38 42

2 2 1 1

57 50 58 56

2 2 3 1

Female 0 1000 5000 10,000

13 14 10 8

13.3 12.9 14.3 13.8

5.04 3.58 4.84 3.05

41 41 42 41

6.03 5.85 6.41 5.89

1.38 1.69 0.76 1-39

6"8 5.9 5.3 4.0*

38 43 43 35

3 2 2 3

57 52 53 60

2 3 2 2

Hb --- Haemoglobin MetHb = Methaemoglobin PCV = Packed cell volume RBC ----Red blood cells R e t i c s = Reticulocytes N = Neutrophils E = Eosinophils L = Lymphocytes M = Monocytes Values are means for 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 ; ***P < 0.00 I. Basophils did not constitute more than 0"5 ~ of the leucocytes in any group and inclusions were not seen in the erythrocytes.

LONG-TERM FEEDING STUDY ON BLACK PN IN RATS

23

Table 5. Results of biochemical analyses of serum and urine from rats fed dietary levels of 0-10,000 ppm

Black P N for 2 yr Serum Dietary level (ppm)

No. of rats examined

Urea (mg/100 ml)

GOT (IU)

GPT (IU)

Urinary GOT (IU)

47.0 43.5 48.5 45.2

19.0 16.3 13.8 15.7

5.7 4.2 5.5 5.7

47.0 41"7 47.9 40.5

14"0 18.0 17"0 18"3

5-4 3.7 5-6 5"0

Males 0 I000 5000 10,000

5 9 6 11

23.0 35.0 42"5 20"0

0 1000 5000 10,000

13 14 10 8

17.0 17-3 16"5 16"6

Females

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. Table 6. Results of renal concentration tests in rats fed dietary levels of O-lO,O00 ppm Black P N for 1 and 2 yr Sex and dietary level (ppm)

No. of rats examined

Specific gravity 0-6 hr

Volume (ml)

16-20 hr

0-6 hr

16-20 hr

Wk 52 Male 0 1000 5000 I0,000

6 6 6 6

1.058 1.056 1.062 1.055

1.087 1.088 !.074 1.067

3.2 2.9 2-4 3.5

1.5 1.0 0-6 0.4

Female 0 1000 5000 10,000

6 6 6 6

1"036 1.043 1"046 1-041

1"072 1'084 1'088 1-082

2-7 3"6 3' 1 3"3

0'9 0'7 0.7 0-5

Wk 104 Male 0 1000 5000 10,000

5 9 6 11

1.031 1.029 1.021 1-028

1.039 1.026 1.025 1-036

5.1 5.1 6.9 5.5

2-6 3.6 3.9 3.7

Female 0 1000 5000 I 0,000

13 14 10 8

1.027 1.022 1.021 1.030

1"029 1'036 ---

4"8 3-7 3"6 3"6

2-1 I "2 ---

The 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.

5 9 6 11

13 14 10 8

5 9 6 11

13 14 10 8

Male 0 I000 5000 10,000

Female 0 1000 5000 10,000

Male 0 1000 5000 10,000

Female 0 1000 5000 10,000

0.45 0.41 0.43 0.43

0.32 0.35 0.37 0.37

1.75 1'76 1.73 1-76

1.99 1.88 1"96 1.95

Brain

0-40 0.34 0.35 0.34

0.38 0.45 0.41 0.42

1.64 1"56 1.53 1.54

2.32 2.38 2.12 2.16

Heart

Kidneys

0.70 0.81 0.81 0.74

1-15 1.16 1.15 1.22 2'98 2.74 2'94 3'02

4.68 5.94 5"86 6'34

Absolute organ weights (g)

Spleen

79 85 74 80

114 94 88 92

Adrenalst

3.27 3.13 3.03 2.87

2.47 3-08" 2.91"** 3.02* 0-20 0.19 0.18 0-18

0'19 0"22 0-22 0"24 0.88 0'64 0.66 0-68

0"78 1"18 1-14 1.26

23 19 25 21

19 18 16 16

Relative organ weights (g/lO0 g body weight)

13-16 14.07 14-18 14.54

14.84 16.01 15'52 15"50

Liver

Organ weights

30 25 27 28

0.62 0.60 0"66 0-65

117 108 106 126

3.77 3.22 3.83 3"57

Gonads +

6-3 5.2 4"4 4"5

2"5 2.5 2-5 2"8

20 21 22 20

15 14 13 15

Pituitaryt

10'5 8"1 8"4 8"3

7.1 7.9 7"3 6"5

38 31 30 35

43 43 38 48

Thyroid'["

377 438 441 443

607 529 533 524

Terminal body weight (g)

tAbsolute and relative weights of this organ are expressed in mg and mg/100 g body weight respectively. +Absolute and relative weights of female gonads are expressed in mg and mg/100 g body weight respectively. Values are means for the numbers of animals shown and those marked with asterisks differ significantly (Student's t test) from those of controls: *P<0-05; ***P< 0.001.

No. of rats examined

Sex and dietary level (ppm)

Table 7. Absolute and relative organ ,'eights o f rats fed dietary levels of O-lO,OOOppm Black P N fi)r 2 yr

t~

O > 7:

>

r~

>

"~

> Z 7.

>

.~

" "~

4~

i,o

LONG-TERM FEEDING STUDY ON BLACK PN IN RATS

25

Table 8. Incidence of histological findings (excluding turnouts) in rats fed dietary levels of 0-10,000 ppm Black P N for up to 2 yr No. of animals affected Males fed levels (ppm) of Organ and histological change

No. of rats examined...

Lung Chronic inflammatory cell infiltration Kidney Chronic rat nephropathy Pancreas Chronic inflammatory cell infiltration Liver Chronic inflammatory cell infiltration of portal tract Testes Atrophy

Females fed levels (ppm) of

0

1000

5000

10,000

0

1000

5000

10,000

21

23

22

21

22

22

23

24

3

8

7

5

3

4

5

5

17

16

15

12

14

9

12

5

1

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

1

2

.

.

.

.

h a e m a t o l o g i c a l results as a w h o le show t h a t Black P N has no u n t o w a r d effect on the h a e m a t o p o i e t i c system. Wh i l e the absolute weight o f the livers was the same in c o n t r o l an d e x p e r i m e n t a l rats, the relative liver weight was h i g h e r in m a l e an i m al s fed diets c o n t a i n i n g Black PN . T h e degree o f e n l a r g e m e n t was n o t related to dose b u t a p p e a r e d to be due to the l o w er ed b o d y Table 9. Incidence of tumours in rats fed dietary levels of O-lO,O00 ppm Black P N for up to 2 yr No of animals with tumours Males fed levels (ppm) of Organ and type of tumour

No. of rats examined...

Mammary gland Adenoma Carcinoma Pancreas Islet cell adenoma Thyroid Adenoma Carcinoma Ovary Thecoma Adrenal Cortical adenoma Subcutaneous tissue Fibroma

Females fed levels (ppm) of

0

1000

5000

10,000

0

1000

5000

10,000

21

23

22

21

22

22

23

24

1

1

2

0

0

0

0

0

9 0

14 0

14 1

10 0

0

1

0

1

I

0

0

0

0 0

0 0

0 0

0 1

0 0

2 0

1 0

0 0

0

0

0

1

0

0

0

1

0

0

0

0

0

0

1

0

0

0

0

0

26

I . F . GAUNT, F. M. B. CARPANINI, P. GRASSO, IDA S. KISS and s. D. GANGOLLI

weight of treated rats compared with those o f controls. The marked variability of body weight generally found in 2-yr old male rats makes it doubtful whether, in fact, relative organ weights determined at this time can be relied upon as an index of potential toxicity. No differences were seen in the relative liver weights of females at 2 yr or in either sex at 3 months, as shown by Gaunt et al. (1967). With the exception of mammary adenomas, tumours were rarely seen in this study. The incidence of mammary adenoma was not related to dosage and these tumours are known to occur frequently with the strain o f rat used (Gaunt et al. 1972; Grasso et al. 1969). The single carcinoma of the mammary tissue was found in a female rat fed a diet containing 5000 ppm Black PN, but no similar lesions were found at twice this dosage level. Mammary carcinoma occurs spontaneously in rats and Sharratt, Frazer & Forbes (1964) found a 1.2 ~o incidence of mammary carcinoma in a group of 750 rats. The one carcinoma of the thyroid found in a treated animal in the present study was again considered to be toxicologically insignificant since these tumours occur spontaneously in experimental rats (Snell, 1967; Russfield, 1967). The benign tumours occurred with a low frequency and the incidence was not related to dosage. We conclude that Black PN does not exert a carcinogenic effect when fed at levels up to 10,000 ppm o f the diet (approximately equivalent to 500 mg/kg/day) and since no findings could be attributed to treatment with Black PN in this study the nountoward-effect level is considered to be 10,000 ppm. This level is similar to that found by Gaunt et al. (1967) in a short-term study of Black P N but is somewhat higher than the level found by Gaunt et al. (1969) in the pig. In the latter species, the pathological finding was attributed to the effect on the gastro-intestinal tract o f the local high concentrations of the colouring which resulted from the fact that pigs consume the whole daily dose in one short period rather than in 6-8 small doses overnight as is the case in the rat. It is difficult to assess the actual intake of food colourings by man, but it has been estimated that the total intake of this colouring in confectionery is in the region of 7 mg/day (0.12 mg/kg/day) in the US (Food Technology, Champaign 1968). If, on the other hand, it is assumed that Black PN is used at a level of 250 ppm in all sugar confectionery, of which 40 g/day is consumed, the intake of colouring would be 10 rag/day (0.17 mg/kg/day). Although it is likely that both these figures are over-estimates, the no-effect level found in the present study is still 1800-2400 times greater. Acknowledgements--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.

REFERENCES British Standards Institution (1968). Specification for Black PN for Use in Foodstuffs. BS 4354:1968.

Deutsche Forschungsgemeinschaft--Farbstoff-Kommission (1957). Mitteilung 6, 2. Auflage. Toxikologische Daten yon Farbstoffen und ihre Zulassung fiir Lebensmittel in verschiedenen 1_2indern. p. 58. Franz Steiner Verlag GmbH, Wiesbaden. Food Standards Committee (1964). Report on Colouring Matters. HMSO, London. Food Technology, Champaign (1968). Guidelines for good manufacturing practice: Use of certified FD&C colors in food. ibid 22, 946. Gaunt, I. F., Brantom, P. G., Grasso, P., Creasey, Margaret & Gangolli, S. D. (1972). Long-term feeding study on Chocolate Brown FB in rats. Fd Cosmet. Toxicol. 10, 3. Gaunt, I. F., Colley, J., Creasey, Margaret & Grasso, P. (1969). Short-term toxicity of Black PN in pigs. Fd Cosmet. Toxicol. 7, 557. Gaunt, I. F., Farmer, Madge, Grasso, P. & Gangolli, S. D. (1967). Acute (mouse and rat) and short-term (rat) toxicity studies on Black PN. Fd Cosmet. Toxicol. 5, 171.

LONG-TERM FEEDING STUDY ON BLACK PN IN RATS

27

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 F A O / W H O 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. 14lid Hlth Org. 309. Karmen, A. (1955). A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood serum. J. clin. Invest. 34, 131. Marsh, W. H., Eingerhut, B. & Miller, 14. (1965). Automated and manual direct methods for the determination of blood urea. Clin. Chem. 11, 624. Russfield, Agnes B. (1967). Pathology of the endocrine glands, ovary and testis of rats and mice. In Pathology of Laboratory Rats and Mice. p. 391. Edited by E. Cotchin and F. J. C. Roe. Blackwell Scientific Publications, Oxford. Ryan, A. J. & Welling, P. G. (1970). The metabolism and excretion of Black PN in the rat and man. Fd Cosmet. ToxicoL 8, 487. Sharratt, M., Frazer, A. C. & Forbes, O. C. (1964). Study of the biological effects of benzoyl peroxide. Fd Cosmet. Toxicol. 2, 527. Snell, Katharine C. (1967). Renal disease of the rat. In Pathology of Laboratory Rats and Mice. Edited by E. Cotchin and F. J. C. Roe. p, 105. Blackwell Scientific Publications, Oxford. Sunderman, F. W. & Sunderman, F. W. Jr. (1960). Hemoglobin, Its Precursors and Metabolites. p. 53. Lippincott, Philadelphia, USA. Walker, R. (1968). Intestinal Degradation of Azo Food Colours with Particular Reference to Brown FK. Ph.D. Thesis, University of Reading, Reading, Berks., England.

Etude nutritionnelle ~ long terme du Noir P N chez le rat R~um6----Deux groupes de jeunes rats sevr6s, 24 m~.les et 24 femelles, ont requ pendant deux ans du Noir PN h raison de 0 (animaux t6moins), 1000, 5000 ou 10 000 ppm du r~gime. Aucun effet imputable au traitement n'a 6t6 observ6 sous le rapport de la mortalit6, du gain de poids, de la consommation de nourriture, de l'h6matologie, de la chimie du s6rum, des tests deconcentration r6nale, du poids des organes et de la fr~.quence des ph6nom6nes pathologiques, tumeurs comprises. Le Noir PN n'a manifest6 aucun pouvoir canc6rig~ne et son seuil d'indiff6rence est de 10 000 ppm (environ 500 mg/kg/jour).

Langzeitiger Verfiitterungsversuch mit Black P N an Ratten Zusammenfassung--Gruppen von 24 m~innlichen und 24 weiblichen abgesetzten Ratten wurden 2 Jahre lang mit Futter geffittert, das 0 (Kontrolle), 1000, 5000 oder 10 000 ppm Black PN enthielt. Es wurden keine dieser Verabreichung zuzuschreibenden Erscheinungen bei der Mortalit~it, Ktirpergewichtszunahme, dem Futterverbrauch, der H~natologie, Serumchemie, NierenKonzentrationstest, Organgewichten oder der H~iufigkeit pathologischer Befunde, einschliesslich Tumoren, festgestellt. Eine carcinogene Wirkung wurde bei Black PN nicht gefunden, und die yon sch~idlichen Wirkungen freie Konzentration war 10 000 ppm (etwa 500 mg/kg/Tag).