Long-term experiment with canned meat treated with sodium nitrite and glucono-δ-lactone in rats

Fd Cosmet. Toxicol. Vol. 10, pp. 475--488. Pergamon Press 1972. Printed in Great Britain

Long-term Experiment with Canned Meat Treated with Sodium Nitrite and Glucono-6-1actone in Rats M. J. VAN LOGTEN, ENGELINA M. DEN TONKELAAR, R. KROES, JOHANNA M. BERKVENS and G. J. VAN ESCH Laboratory of Toxicology and Laboratory of Pathology, National htstitute of Public Health, Bilthoven, The Netherlands (Received 27 March 1972) Abstract--A long-term toxicity experiment was carried out in rats fed a diet containing 40% canned meat treated with either 0-5 or 0.02 % sodium nitrite with or without glucono-8-1actone. This experiment, in which special attention was paid to the detection of a possible carcinogenic effect, was designed to study the possible formation of nitrosamines from nitrite and the secondary amines present in meat, under the conditions of an acid pH and pasteurization. The parameters studied included haematology, clinical biochemistry, histopathology, a-foetoprotein in the serum and the DNA content of the liver cell nuclei. There was no evidence of any preneoplastic change or tumour formation that could be attributed to the feeding of this canned meat.

INTRODUCTION Most N-nitrosamines are known to be powerful carcinogens. Since the discovery by Magee & Barnes (1956) of the carcinogenic activity of dimethylnitrosamine, numerous N-nitroso compounds have been tested for their carcinogenic activity. Druckrey, Preussmann, Ivankovic & Schm~ihl (1967) have presented a detailed review of studies they carried out in rats on the relation between the structure and carcinogenicity of 65 nitrosamines. Striking organotropic effects were observed. The nitrosamines tested, either by the oral or sc route, induced tumours in the liver, kidneys, oesophagus, nasal cavity, stomach, lungs, urinary bladder and central nervous system. The structure of the compound, the dose level and the route of administration often determined the site and type of tumour that developed. The carcinogenic action of N-nitroso compounds has also been reviewed by Magee & Schoental (1964) and Magee & Barnes (1967). F r o m the results of Schm~hl & Osswald (1967) it is clear that nitrosamines induce tumours in many animal species. Following the work of Ender, Havre, Helgebostad, Koppang, Madsen & Ceh (1964), the suspicion arose that nitrite could combine with secondary or tertiary amines present in fish and meat to form nitrosamines. In addition, many experiments were carried out on the formation of nitrosamines in the stomach after simultaneous administration of high levels of nitrite and secondary amines (Sander, Schweinsberg & Menz, 1968). Sander (1971) and Sander & Btirkle (1969) reported the induction of malignant turnouts by the simultaneous feeding of nitrite and certain basic secondary amines, such as N-methylbenzylamine and N-methylaniline. In these cases the nitrosamines were assumed to have been formed in the stomach and not in the diet. Sander (1971) also surveyed the possible occurrence of nitrite and secondary amines in the human environment. In a recent paper by Magee (1971), the possible health hazards of nitrosamines are summarized in relation to the occurrence of these substances in food. In the production of 475

476

M . J . VAN LOGTEN~ E. M. DEN TONKELAAR, R. KROES et

al.

several meat products, nitrite is added as a preservative and the meat is subsequently pasteurized or sterilized. Sometimes glucono-8-1actone ( G D L ) is also added to lower the pH. Heat and a low p H are favourable conditions for the formation of nitrosamines from nitrite and secondary amines. On the other hand the haemoglobin or myoglobin present in the meat serves to neutralize the dangerous properties of the N O + ion by reaction with Fe 2+. Therefore it was considered to be advisable to carry out a long-term toxicity test, in which special attention would be given to the possible induction of tumours by the ingestion of meat treated in this way. EXPERIMENTAL Diets Preparation o f canned meats. The meat for the diets was prepared by the Central Institute for Nutrition and Food Research T N O at Zeist, and consisted of 28 70 beef, 24 70 pork meat, 28 70 bacon, 12 70 water, 4 ~ starch, 2 70 NaCI and 0.3 ~ polyphosphate. In addition, the meat for the test diets was treated with 0.5 70 NaNO2, with 0.5 ~ NaNO2 together with I Yo G D L , with 1 70 G D L alone, or with 0.02 ~ NaNO2 and 1 ~ G D L . The components of the basic meat mixture were mixed and stored in charges of 25 kg in a deep-freeze at --40°C. Before the preparation of the diets, this meat was partially defrosted for one night at 15°C. Thereafter the different components were cut and thoroughly mixed. During this process the nitrite and/or G D L were added. The meat-dough was then canned in tins of 200 g and pasteurized at 80°C for 70 min. During the heating o f tins that contained 0.5 NaNO2 and 1 70 G D L , an effective pressure of 1"25 atm. was used to compensate for the internal pressure caused by a reaction between the nitrite and G D L . The prepared canned meat was stored at --20°C. For the whole experiment five batches each consisting of four charges o f 25 kg and one batch consisting o f two charges of 25 kg were prepared. The batches were prepared every 4 months. Analysis o f prepared meats. The content of nitrite, fat and water and the p H were determined on each batch after pasteurization (Table I). The average protein content of the meat used was 12~o. It was originally planned to analyse all batches for the presence of nitrosamines, but with the methods available at that time nitrosamine could not be determined in concentrations below 1 ppm. For this reason samples of the different batches were kept. Recently samples of the last batch were sent to Dr. H. Egan of the Laboratory of the

Table 1. Average pH and nitrite, fat and water content of test and control meats after pasteurization Content of Sample

pH

NaNO2 (ppm)

Fat (70)

Water (70)

Untreated meat Meat + 0"570 NaNO~ Meat + GDL Meat + 0"5~ NaNO2 + GDL Meat + 0-02~NaNO2 + GDL

6"42 4- 0"14 6-62 4- 0-19 5.19 4- 0.10 5.71 4-0.11 5.27 4- 0.12

0"4 4- 0.5 4004 4- 308 0-4 4- 0'5 1824 4- 296 9 4- 3

28-6 :k 1"I 28"4 4- 0.7 28'2 4- 1.0 28"6 4- 1.1 26"3 4- 0"9

54"4 4- 0"5 54"6 4- 0'4 53.9 4- 1.0 53-4 4- 0.4 54"1 4- 0"5

GDL = Glucono-8-1actone Each value represents the mean of six batches.

FEEDING STUDY ON NITRITE-TREATED MEAT

477

Government Chemist, London, for analysis. According to the preliminary results, no nitrosamines were detected in the control meat or in the meat treated with 0-02 % NaNO2 and 1% GDL. Meat treated with 0-5 % NaNO2 contained 30 ppb* dimethylnitrosamine and < 2 ppb diethylnitrosamine, the presence of both nitrosamines being confirmed in this sample by mass spectrometry. Meat treated only with 1% GDL contained < 1 ppb dimethyl- and 5 ppb diethylnitrosamine and < 1 ppb N-nitrosopyrrolidine, while that treated with 0.5 ~ NaNO2 and 1% GDL contained 40 ppb dimethyl- and 14 ppb diethylnitrosamine, 4 ppb N-nitrosopiperidine and 4 ppb N-nitrosopyrrolidine. Compounding of diets. At the National Institute of Public Health, the canned meats were defrosted weekly and mixed with a standard diet (Muracon-SSP-Tox) in a weight ratio of 40 ~o meat in SSP-Tox. The various diets were stored under refrigeration until they were given to the animals 3 times/wk. The animal food, Muracon-SSP-Tox, is specially compounded for our laboratory by the firm Trouw and Co., Putten, The Netherlands, and consists of casein, soya protein, maize starch, glucose, cellulose, soya oil, lard, a salt mixture and vitamins.

Animals Male and female SPF-derived Wistar rats were obtained from the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands. They were housed in wire cages, two animals to a cage. Drinking water was given ad lib. At the beginning of the experiment food was also given ad lib., but when the weight gain became too high the animals were put on rations (about 20 g/day).

Experimental design and conduct Six groups, each of 30 male and 30 female rats, were fed from just after weaning either on the standard SSP-Tox diet (control group), on a diet containing 40 % meat (meat-control group) or on a diet containing 40 ~ meat treated either with 0-5 % NaNO2, with 1 ~o GDL, with 0.5 % NaNOz and 1% GDL or with 0.02 % NaNO2 and 1 ~ GDL. The animals were weighed weekly during the first 13 wk and every 4 wk thereafter. Food intake was recorded only in wk l, 2, 5, 9 and 13. Blood samples for haematological investigations were taken from ten animals in each group after 12, 24, 37, 51, 66, 78 and 91 wk, the haemoglobin level, packed cell volume, numbers of erythrocytes and total and differential leucocytes were recorded and the mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated. The bromsulphthalein (BSP) retention test was used as an index of liver function together with determinations of serum glutamic-pyruvic transaminase (SGPT) activity. These tests were carried out at wk 13 in five males and at wk 26 in five females from each group. SGPT, alkaline phosphatase and urea were determined after 9, 12 and 16 months in blood from male and female rats. Throughout the study, animals were killed if they became moribund. Of the rats dying in this way when about 2 yr old, six from each group were used for determinations of the activities of the hepatic enzymes, aniline hydroxylase, aminopyrine demethylase and glucose 6-phosphatase. All animals were inspected regularly for the presence of tumours. After 29 months the experiment was terminated, the remaining animals being killed with carbon dioxide. * b = 109 throughout this paper.

478

M.J. VAN LOGTEN, E. M. DEN TONKELAAR, R. KROES et al.

All animals that had died or were killed were autopsied and all organs were fixed in buffered 10 ~ formalin or Bouin solution for histopathological examination. The following organs and tissues were studied: liver, kidneys, heart, lungs, brain, spleen, pituitary gland, thyroid, thymus (if any), pancreas, adrenals, testes, ovaries, prostate, uterus, lymph nodes, salivary glands, gastro-intestinal tract (six levels), urinary bladder, spinal cord (three levels), peripheral nerves, skeletal muscles, skin and m a m m a r y gland. A t the end o f the experiment smears were made f r o m the livers o f two, three or four rats in each group, except in that receiving 0.5 ~o nitrite and G D L , f r o m which seven animals were taken. The smears were stained according to the Feulgen technique for D N A . The a m o u n t o f D N A / c e l l nucleus was measured microspectrophotometrically with the Zeiss Cytoscan. All animals alive at 29 m o n t h s were bled and their serum was tested for the presence o f a-foetoprotein by double diffusion in 0.8 ~ agarose. Statistical methods Statistical analysis was carried out on all parameters except food intake, for which no individual data were available. The statistical calculations were carried out according to the Student's t test. RESULTS Growth, f o o d intake and mortality The mean b o d y weights for each g r o u p recorded at wk 6, 13, 26, 52, 78 and 104 are given in Table 2. These show that a diet containing 40 ~o meat gave a higher b o d y weight than a standard animal diet. However, the groups receiving meat treated with N a N O 2 (0.5 ~ ) , with or without G D L , generally showed a lower b o d y weight than the meat-control group. Table 2. Mean body weight of rats fed diets containing 40 ~ meat treated with nitrite attd/or GDL Body weight (g) at wk Diet

0

Control Meat control Meat + 0-5 ~ NaNO2 Meat + GDL Meat + 0"5~ NaNO2 + GDL Meat + 0"02~o NaNO2 + GDL

48 51 50 48 49 49

Control Meat control Meat + 0.5~ NaNO2 Meat + GDL Meat + 0.5~ NaNO2 + GDL Meat + 0-02~ NaNO2 + GDL

46 45 46 46 46 47

6

13

26

52

78

104

366*** 406 367*** 389 344*** 383*

436*** 487 438*** 462 421"** 457*

539*** 632 566*** 608 535*** 594*

576*** 650 583** 654 578*** 638

579 622 582 651 589 591

221"* 240 228 241 209*** 236

249*** 278 264 278 231'** 272

313"** 379 355 369 295*** 363

354** 409 378 392 335*** 412

330 381 387 434 348 395

Males

280** 297 268*** 280* 251"** 280* Females

181 186 179 190 167"** 186

GDL = Glucono-6-1actone Values marked with asterisks differ significantly (Student's t test) from those of the meat-control group: *P < 0.05; **P < 0"01; ***P < 0"001.

FEEDING STUDY ON NITRITE-TREATED MEAT

479

The food intake was estimated only during the first 13 wk o f the experiment. It was clear hat all animals on the meat diets had a higher food intake than the control animals on a standard laborabory diet, but there were no differences between the meat-control group and the groups receiving meat treated with NaNO2 and/or GDL. The behaviour and appearance of the rats remained good during the first 18 months. Thereafter the mortality increased rapidly in all groups receiving meat (Table 3). Neither NaNO2 nor G D L had any obvious influence on mortality, however. Table 3. Cumulative mortality of rats fed diets containing 40*/. meat treated with nitrite

and/or GDL Initial group size

Diet

Mortality at m o n t h 12

15

18

21

24

27

29

1 0 0 1 0 0

3 1 1 1 0 1

4 2 4 3 2 3

5 9 9 6 5 13

14 17 14 18 15 18

19 27 21 25 19 24

24 30 26 25 22 28

0 1 2 1 1 0

0 2 2 1 3 1

0 5 4 3 3 4

4 10 8 8 5 8

9 23 18 18 14 17

19 28 25 23 23 26

24 30 26 24 27 27

Males

Control Meat control Meat + 0'5~o NaNO2 Meat + GDL Meat + 0-5 ~ NaNO2 + GDL Meat + 0-02~ NaNO2 + GDL

30 30 30 30 30 30 Females

Control Meat control Meat + 0.5 ~o NaNO2 Meat + GDL Meat + 0.5 ~o NaNO2 + GDL Meat + 0.02~ NaNO2 -t- GDL

30 30 30 30 30 30

G D L = Glucono-8-1actone

Haematology In Table 4 only the results for haemoglobin, erythrocytes, total leucocytes and percentages of neutrophils and lymphocytes after 12, 51 and 91 wk are given. No differences, or only incidental ones, were found in the packed cell volume, the numbers of monocytes and eosinophils and the MCV, M C H and MCHC. The number of erythrocytes was lower in the groups receiving nitrite. The haemoglobin content of the blood also showed a tendency towards a lower value in these groups, but the effect was less clear than with the red cell count. Both changes may have been due to the addition of nitrite to the meat. No differences were observed in the numbers of leucocytes. As the animals grew older there was a decrease in the relative number o f lymphocytes and an increase in that of neutrophils.

Clinical biochemistry The results of the BSP-retention and SGPT determinations carried out at 3 and 6 months are given in Table 5, while Table 6 presents the results o f SGPT, alkaline phosphatase and blood-urea determinations at 9, 12 and 16 months. It is well-known that nitrosamines cause liver damage and if sufficient nitrosamines had been formed in the treated meat, one would have expected abnormalities to have been detected in these liver-function tests (den Tonkelaar & Kroes, 1971). However, no abnormal values were found in the group receiving meat treated with 0.5 ~o NaNO2 and GDL. The results after 9 months showed a tendency F.C.T. | 0 / 4 ~ C

480

M.J. VAN LOGTEN~ E. M. DEN TONKELAAR~ R. KROES et al.

Table 4. Haematological studies in rats fed diets containing 40 ~ meat treated with nitrite and[or GDL Haematological values in Males at wk Diet

12

51

Females at wk 91

12

51

91

15-2" 14.7 14-6 14.9 14'6 15.1"

15"5 15.4 14.7" 15"3 15.1 15.5

14-4 14'7 13.8 15"8 14'7 15"3

7.5 7-3 7.1 7.3 6.9 7.4

7.3 7"0 6.6 6.9 6-5 6.7

6,9 6.7 6-4 7.6* 6.9 7-1

Haemoglobin (g/100 ml) Control Meat control Meat + 0'5~0 NaNO2 Meat + GDL Meat q- 0"5% NaNO2 h- GDL Meat + 0"02~ NaNO2 + GDL

15"2 15.5 14-1" 15"4 14-8" 14'9

15.4 15.4 14-9 15.7 15.2 15.4

15"3 14.6 13"5 14.6 14.0 14"1

Control Meat control Meat + 0.5 ~0 NaNO2 Meat + GDL Meat + 0.5 ~ NaNO2 + GDL Meat q- 0.02~ NaNO2 + GDL

7"7 7-8 7-2* 7.8 7.4* 7.6

Control Meat control Meat + 0"5~o NaNO2 Meat + GDL Meat + 0.5~ NaNO2 + GDL Meat + 0"02~o NaNO2 q- GDL

Total leucocytes (103/mln 3) 12'4"* 8"8 8.7 10.9 9.1 8'9 12-4 10.1 9.7 11.2 9-1 10.7 11.3 8-5 8-9 11.3 10-8" 10.3'

10"1 9-4 9-7 9.2 10.7 9-0

6"5 6.6 7.5* 7.6 7.5* 7.6*

7.1 6-4 7.1 7.9* 8.0* 6.6

Control Meat control Meat -k 0.5 ~o NaNO2 Meat -I- GDL Meat -k 0.5 ~o NaNO2 q- GDL Meat -k 0.02~ NaNO2 -k GDL

Neutrophils ( % of leucocytes) 9"9 15'2 23.6 11.7 15.4 25.8 11.1 15.5 23.7 10-7 15.4 31.0 10-7 17.6 28'2 15'5 19.6 30-6

10.7 11.0 11"3 10"3 9.5 8-9

11-5 14-0 13"4 12.0 14.3 11.9

31 '6 27"2 28-7 32"I 23"9 30"2

Control Meat control Meat + 0.5 ~ NaNO2 Meat q- GDL Meat + 0.5% NaNO2 + GDL Meat + 0.02% NaNO2 -I- GDL

86"3 84"1 84-8 83.7 85"5 81.3

86.2 85'9 86.3 85.7 86.9 87.2

83"3 81 "4 82-6 82-7 81.0 82.1

63" 1 67"3 67"3 62.2 72-7 64.0

Erythrocytes (106/mma) 7-7 7.5 7.0** 7.4 7.0* 7.0*

7-6 7"4 7.0 7-1 6.9 7.0

Lymphocytes ( % of leucocytes) 78.3 78-2 79.2 78.7 77.0 75.2

70.5 67.8 71'4 63.8 66.2 63.8

GDL = Glucono-8-1actone Values marked with asterisks differ significantly (Student's t test) from those of the meat-control group: *P < 0.05; **P < 0"01. to a higher alkaline p h o s p h a t a s e value in the g r o u p s r e c e w i n g 0.5 ~ N a N O 2 . T h e increases b e c a m e less m a r k e d at later stages a n d especially after 16 m o n t h s , a l t h o u g h m o r e a n i m a l s f r o m these g r o u p s were used f o r the later estimations. W h e n the e x p e r i m e n t h a d lasted for a b o u t 2 yr, six m o r i b u n d rats f r o m each g r o u p (three males a n d three females or two males a n d f o u r females) were killed a n d used to study the aniline-hydroxylase, a m i n o p y r i n e d e m e t h y l a s e an d g l u c o s e - 6 - p h o s p h a t a s e activity in the liver. I n h i b i t i o n o f these en zy m es is

FEEDING STUDY ON NITRITE-TREATED MEAT

481

probably a more sensitive indicator of liver damage than the liver function tests performed on serum. There was a wide variation in the values and only a few animals were used from each group but the results did not show any tendency towards inhibition of enzyme activity in the groups receiving 0.5 ~ NaNO2 with or without GDL. Table 5. Liver-function tests hz rats fed for 3 or 6 months on diets containing 4 0 ~ meat treated with nitrite and~or G DL

Mean values in Males after 3 months Diet Control Meat control Meat + 0-5~o NaNO2 Meat + G D L Meat + 0 . 5 ~ NaNO2 + GDL Meat + 0 . 0 2 ~ NaNOz + G D L

BSP (m-moles/litre) 14.9 15" 1 15-5 15.5 10.0 16-6

SGPT (U/litre) 16 15 15 15 15 18

Females after 6 months BSP SGPT (m-moles/litre) (U/litre) 11"7 13'9 11.0 14.5 14.1 15.8

11 11 9 10 8 10

BSP = Bromsulphthalein GDL = Glucono-8-1actone SGPT = Serum glutamic-pyruvic transaminase The serum of each of the five rats/group was used for both determinations. No statistically significant differences were found with Student's t test. During the final months of the experiment a number of animals showed oedema. Therefore in all animals that were killed in a moribund condition or at the end of the experiment, the blood urea was determined. The results are given in Table 7. Although a number of animals showed values that were moderately high (7-1-9.9 m-moles/litre) or high ( > 10 mmoles/litre), these were randomly distributed among the groups and no clear effect of the various diets on kidney function could be demonstrated.

Histopathology All animals that died or were killed were studied histopathologically except for a few in which this was prevented by advanced autolysis. Gross observations revealed no differences between the various groups. The many histological abnormalities observed in the organs and tissues were scattered throughout all the groups and were considered to be abnormalities usually present in the strain of rat used. The most c o m m o n abnormality was glomerulonephrosis sometimes associated with uraemia and metastatic calcification in several organs, especially the heart. Pronounced hyperplasia of the parathyroids was also found in the same animals. Tables 8 and 9 list only the tumours found in rats that survived for 18 months or more. The incidence of spontaneous tumours was considerable. In the "target organs" for nitrosamine carcinogenesis, notably the lungs, oesophagus, gastrointestinal tract, liver, kidney, urinary bladder and central nervous system, there were no tumours that could be related to the administration of meat treated with NaNO2 and/or G D L (Table 10).

10 5 l0 5 l0 5

10 5 10 5 10 5

Control Meat control Meat + 0.5° NaNO2 Meat + G D L Meat + 0 - 5 ~ NaNO2 -}- G D L Meat + 0-02% NaNO2 + GDL

Control Meat control Meat + 0 . 5 ~ NaNO2 Meat + GDL Meat + 0 ' 5 ~ NaNO2 + G D L Meat + 0'02~o NaNO2 + GDL

14 23 15 16 13 17

10 10 11 18 9 11

10 l0 9 15 9 13

SGPT (U/litre)

7.0 5.5 6'5 5'8 6-8 6'0

5.5* 4"2 4.8 5.8* 4.8 5'2

5.8 5"0 4-6 6.5 4.8 7.2

12 months 38 42 55 43 43 40

16 months 33 33 40 30 37 35

Urea (m-moles/litre)

9 months 52 63 77 68 78 60

Alk. Pase (U/litre)

Males

12 17 13 15 18 18

11 * 15 12" 20 14 15

9 13 9 15 14" I1

SGPT (U/litre)

32 37 33 37 40 32

47 48 67 58 65 48

53 72 72 63 78 53

Alk. Pase (U/litre)

Females

5.9 5'2 4"4 7.8 5-6 4'6*

4"8 4-7 4.3 5"8 5"0 5-3

6"7 6.0 5-8 5"7 5"7 5"7

Urea (m-moles/litre)

Alk. Pase = Alkaline phosphatase G D L = Glucono-G-lactone SGPT = Serum glutamic-pyruvic transaminase Values marked with an asterisk differ significantly (Student's t test) from those of the meat-control group: *P < 0"05.

5 5 5 5 5 5

Control Meat control Meat + 0'5% NaNO2 Meat + G D L Meat + 0-5 % NaNO2 + G D L Meat + 0-02~ NaNOz + G D L

Diet

No. of animals tested

Mean values in

Table 6. Clinical chemistry of rats fed diets containing 40 ~ meat with nitrite and[or GDL

7~

r-" > >

Z ,.-1 © Z

rrl

ra

rn

oo t,a

FEEDING STUDY ON NITRITE-TREATED MEAT

483

Table 7. Terminal blood-urea values of rats fed diets containing 40 % meat treated with nitrite and/or GDL and killed during the last months or at the end of the study (frequency table)

No. of rats tested

Diet

No. of rats with blood-urea levels (m-moles/litre) of <7

7' 1-9.9

> 10

9 5 5 5 9 2

3 2 3 3 3 -

5 1 2 1 4 -

1 2 1 2 2

10 3 6 7 6 6

3 1 5 4 4 3

4 2 1 3 1 1

3 l 2

Males

Control Meat control Meat + 0.5% NaNO2 Meat + GDL Meat + 0'5~/ooNaNO2 + GDL Meat + 0-02% NaNOz + GDL Females

Control Meat control Meat + 0"5% NaNO2 Meat + GDL Meat + 0"5 % NaNO2 + GDL Meat + 0"02+ NaNO2 + GDL

GDL = Glucono-8-1actone D N A and a-foetoprotein determinations

M e a s u r e m e n t o f the D N A c o n t e n t o f i n d i v i d u a l cell nuclei, used as an i n d i c a t o r o f p r e n e o p l a s t i c changes in the liver, revealed no cells with an a b n o r m a l D N A content. T h e r a t i o between d i p l o i d a n d t e t r a p l o i d cells was n o r m a l a n d o n l y a few o c t a p l o i d cells were found. T h e r e were no differences between the groups. Similarly, no positive r e a c t i o n s were o b t a i n e d when s e r u m samples f r o m all a n i m a l s killed at the end o f the e x p e r i m e n t were tested for a - f o e t o p r o t e i n , the presence o f which m a y indicate the existence o f liver t u m o u r s (Abelev, 1971). DISCUSSION As early as 1963, a l o n g - t e r m e x p e r i m e n t with nitrite was described by D r u c k r e y , Steinhoff, Beuthner, S c h n e i d e r & Kl~irner (1963). T h r e e g e n e r a t i o n s o f rats were given I00 m g N a N O 2 / k g b o d y w e i g h t daily in the d r i n k i n g water. In a d d i t i o n , a n i m a l s f r o m the F I generat i o n were s i m u l t a n e o u s l y given d i e t h y l a m i n e in the diet ( i n c o r p o r a t e d in m i n c e d meat). A m o d e r a t e l y r e d u c e d lifespan in all g r o u p s ( c o m p a r e d with c o n t r o l a n i m a l s f r o m o t h e r e x p e r i m e n t s ) b u t no influence on h a e m o g l o b i n c o n t e n t o r on the n u m b e r o f e r y t h r o c y t e s was observed. A certain a m o u n t (up to 20 ~ ) o f m e t h a e m o g l o b i n was found. H i s t o p a t h o logical e x a m i n a t i o n revealed a few t u m o u r s , b u t these were c o n s i d e r e d to be o f s p o n t a n e o u s origin and no i n d i c a t i o n was o b t a i n e d t h a t nitrite alone, or in c o m b i n a t i o n with d i e t h y l a m i n e , h a d a n y c a r c i n o g e n i c o r t e r a t o g e n i c effect. In o u r experiments, N a N O 2 at levels u p to 0.5 % was i n c o r p o r a t e d in c a n n e d meat, with o r w i t h o u t G D L , before processing. G r o w t h was inhibited in the g r o u p s receiving c a n n e d m e a t with the higher level o f a d d e d nitrite c o m p a r e d with the g r o u p receiving the u n t r e a t e d m e a t diet. T h e h a e m a t o l o g i c a l d a t a suggest t h a t c o n s u m p t i o n o f a m e a t diet c o n t a i n i n g a d d e d nitrite causes a decrease in the n u m b e r o f erythrocytes. T h e h a e m o g l o b i n c o n t e n t o f the b l o o d was n o t affected in a clearly

484

M. J. V A N L O G T E N , E. M. D E N T O N K E L A A R ~ R. KROES e l

a[.

T a b l e 8. Tumour incidence in male rats that survioed for 18 months or more on diets containing

4 0 % meat treated with nitrite and/or G D L Tumour incidence in raLs fed Control diet Organ

No. of animals studied . . .

Lungs Bronchial adenoma Squamous cell carcinoma Liver Bile-duct carcinoma Hyperpla~tic nodules Kidneys Hamartoma Pituitary gland Chromophobic adenoma Thyroid Papillary adenoma Solid cell carcinoma Adrenals Cortical adenoma Medullary phacochromocytoma Pancreas Insuloma Adenocarcinoma of exocrine system Adenoma o f exocrine system Testes Interstitial cell turnout Urinary bladder Papilloma Haemo]lymphopoietic system Leukaemia Abdomen Sarcoma Thorax Mesothelioma Ear-duct gland Squamous cell carcinoma Subcutaneous tissue Fibroma Myxoma Sarcoma

Meat.control Meat + 0.5% diet NaNOj

Meat + GDL

Meat + 0 5% NaNOI + G D L

Meat + 0 ' 0 2 ~ HaNOi + GDL

25

28

26

27

25

26

0 1

0 0

0 0

1 0

0 0

0 0

0 0

0 0

0 0

I 0

0 1

0 0

0

0

0

I

0

0

6

!

2

I

3

4

0 0

0 0

0

1

0

0

1

0

0

0

0 2

0 6

0 6

I 2

I 5

2 2

2 I 0

0 0 0

0 0 0

2 0 0

0 0 I

2 0 I

1

I

2

0

I

0

0

0

0

0

0

I

0

0

0

0

0

I

0

0

I

0

0

0

1

0

0

0

0

0

0

1

0

0

0

1

2 0 0

I 0 I

0 0 2

0 0 I

2 1 0

0 0 0

GDL = Glucono-3-1actone recognizable manner. The results of Musil (1966), who also studied the effect on the blood o f rats of a fairly high nitrite intake over 200 days, point to the same conclusion. The analytical data show that meat treated with 0-5 ~o (5000 ppm) nitrite still contains about 4000 ppm after processing. In the meat treated with 0.5 ~o nitrite and G D L less than 2000 ppm nitrite is left after processing, while in the group treated with 0.02 ~o nitrite and G D L only about 10 ppm remains. Consequently the experiments were performed with final concentrations of free nitrite of about 1600, 750 and 4 ppm. Haematological data revealed changes in the groups actually receiving 1600 and 750 ppm, so that it is likely that some reaction products of nitrite and meat were involved in this phenomenon and not nitrite as such. Since nitrosamines are strong hepatotoxic agents, liver damage could be expected if sufficient nitrosamines were formed. However, liver-function tests (the BSP test and the determination of the activity o f S G P T ) d i d not indicate any such effect. Microsomal liver enzymes were studied as an indicator o f the effect of nitrosamines on the endoplasmic reticulum, but no decrease in the activities of glucose 6-phosphatase, aniline hydroxylase or aminopyrine demethylase was found.

FEEDING

Table

9.

STUDY ON NITRITE-TREATED

MEAT

485

Tumour incidence in female rats that survived for 18 months or more on diets containing 40 % meat treated with nitrite and/or GDL T u m o u r incidence in rats fed

Organ

N o . o f animals studied...

Liver Bile-duct adenoma Hyperplastic nodules Kidney Cortical adenorna Brain Meningioma Astrocytoma Pituitary gland Chromophobic a d e n o m a Eosinophilic a d e n o m a Thyroid Papillary adenoma Adrenal Medullary phaeochromocytoma Pancreas Insuloma A d e n o m a o f exocrine system Uterus Adenocarcinoma M a m m a r y gland Adenoma Adenocarcinoma Intestine Colonic adenocarcinoma Haemo/lymphopoietic system Leukaemia Abdomen Mesothelioma Sarcoma Thorax Sarcoma Ear-duct gland Squarnous cell carcinoma Subcutaneous tissue Fibroma

Control diet

Meat-control diet

Meat + 0"5% NaNO~

Meat + GDL

M e a t + 0"5% NaNO) + GDL

M e a t + 0"02% NaNO~ + G D L

28

25

25

27

27

25

0 1

0 0

0 I

1 0

0 2

0 0

0

1

0

0

1

0

I 0

0 I

0 0

0 0

0 0

0 0

8 0

7 I

8 0

6 0

7 0

9 0

0

0

I

0

0

0

I

0

2

1

0

0

0 0

0 0

I 0

0 0

0 0

0 I

0

0

I

0

0

0

5 0

2 0

3 0

I 0

3 0

4 2

0

0

0

0

0

1

0

1

I

I

2

0

I 0

0 0

0 I

0 0

0 0

0 0

0

I

0

0

0

0

0

0

0

I

0

0

0

1

I

1

0

0

GDL = Glucono-8-1actone

Druckrey et al. (1967) studied the carcinogenic effects of 65 different N-nitroso compounds in rats, with particular reference to the target organs. In general, symmetrical dialkylnitrosamines produced carcinomas of the liver after oral administration. These authors found also that diethylnitrosamine, at lower dose levels, produced not only liver tumours but also tumours of the oesophagus and nasal cavity. The non-symmetrical dialkylnitrosamines generally produced oesophageal tumours but no liver tumours. On the other hand, cyclic nitrosamines like N-nitrosopyrrolidine produced mainly liver tumours. In our own experiments with dimethylnitrosamine (den Tonkelaar & Kroes, 1971) many tumours occurred in the kidneys and lungs in addition to liver turnouts, an observation already reported by Magee & Barnes (1962). Druckrey et al. (1967) also studied the dose-response relationship and found that it followed the general formula dtn = constant, where t is the induction time and d the dosage. This means that a carcinogenic substance present in a diet in a very low concentration shows a very long latent period. Therefore the rats iv the experiment described in the present paper were kept as long as possible.

Total no. o f t u m o u r s . . .

*Hyperplastic nodules.

Lungs Liver Kidneys Brain Oesophagus Intestine U r i n a r y bladder

Organ

N o . o f rats studied...

3

1 1 0 1 0 0 0

53

Control diet

1

0 1 0 0 0 0 0

51

M e a t + 0.5 ~0 NaNO2

G D L = Glucono-G-lactone

2

0 0 1 1 0 0 0

53

Meat-control diet

4

1 2 1 0 0 0 0

54

Meat + GDL

T u m o u r incidence in rats fed

4

0 3* 1 0 0 0 0

52

M e a t + 0"5 ~o NaNO2 + GDL

2

0 0 0 0 0 1 1

51

Meat + 0.02~ NaNO2 + GDL

Table 10. Tumour incidence in "target organs'for nitrosamine carcinogenesis in male and female rats f e d diets containing 40% meat treated with nitrite and[or G DL

©

rn t-

m Z ,.-1

Z .m

0

Z

oo

FEEDING STUDY ON NITRITE-TREATED MEAT

487

N o increased t u m o u r incidence was n o t e d in the g r o u p s receiving m e a t with nitrite a n d / o r G D L . T h e t u m o u r s f o u n d in the o r g a n s t h a t can be c o n s i d e r e d as target o r g a n s for nitros a m i n e carcinogenesis were equally d i s t r i b u t e d a m o n g all groups. In a d d i t i o n , the m e a s u r e m e n t s o f D N A in liver nuclei a n d the results o f the e x a m i n a t i o n o f sera for ct-foetoprotein did n o t give a n y i n d i c a t i o n o f the presence o f p r e n e o p l a s t i c liver changes. P r e l i m i n a r y a n a l y t i c a l d a t a indicate t h a t the m e a t t r e a t e d with 0-5 % nitrite a n d G D L c o n t a i n e d a b o u t 50 p p b nitrosamines. This level c o r r e s p o n d s to 20 p p b in the diet o f the rats or a daily i n t a k e o f 1 ~ g / k g b o d y weight. In the m e a t t r e a t e d with 0,02 ~ nitrite (the n o r m a l level o f use) in c o m b i n a t i o n with G D L , no n i t r o s a m i n e s at all were detected in the prel i m i n a r y analyses. D r u c k r e y et al. (1967) s h o w e d t h a t a daily intake o f 75 p.g diethyln i t r o s a m i n e / k g b o d y w e i g h t i n d u c e d a small n u m b e r o f t u m o u r s in three target organs, the m e a n latent p e r i o d being 840 days. C u r r e n t e x p e r i m e n t s in o u r own l a b o r a t o r y using a s u b m i n i m a l dose level o f a b o u t 12.5 #g pure d i e t h y l n i t r o s a m i n e / k g b o d y w e i g h t / d a y have so far (after 20 m o n t h s ) yielded no t u m o u r s . It is therefore n o t surprising t h a t no t u m o u r s were f o u n d after the feeding o f m e a t t r e a t e d with nitrite a n d / o r G D L , even in the experim e n t a l g r o u p with the unrealistically high dose level o f 0.5 ~o N a N O 2 a n d a l o w e r e d p H . In e v a l u a t i n g the possible h a z a r d s o f a d d i n g nitrite to m e a t p r o d u c t s , it is i m p o r t a n t to r e m e m b e r t h a t at present it seems impossible to d i s c o n t i n u e the use o f nitrite because o f the d a n g e r o f b o t u l i s m that c o u l d follow the o m i s s i o n o f nitrite f r o m certain m e a t p r o d u c t s . Since it is k n o w n (Sander, 1971) t h a t n i t r o s a m i n e s are also f o r m e d in the g a s t r o - i n t e s t i n a l t r a c t f r o m nitrite a n d s e c o n d a r y a n d t e r t i a r y amines, the e v a l u a t i o n o f the risk to h e a l t h f r o m the use o f nitrite in m e a t p r o d u c t s s h o u l d t a k e into a c c o u n t the fact t h a t b o t h g r o u p s o f substances o c c u r widely in f o o d a n d in the general e n v i r o n m e n t . Acknowledgements--We are grateful to Prof. Ir. B. Krol and lr. P. C. Moerman (Central Institute for Nutrition and Food Research TNO, Zeist) for analysing the canned meats, to Dr. P. van Duyn, Dr. M. van der Ploeg, Miss J. Allaart and Mrs. G. Posthuma-Trumpie (University of Leiden) for the measurements of DNA in liver cell nuclei, to Dr. H. Egan and Mr. J. K. Foreman (Laboratory of the Government Chemist, London) for the determination of nitrosamines in the meat, to Dr. P. S. Elias for help with editing, to Dr. P. W. Helleman for the haematological studies and to Mr. T. de ¥ries and co-workers for the care taken in this experiment.

REFERENCES Abelev, G. I. (1971). Alpha-fetoprotein in ontogenesis and its association with malignant turnouts. Adv. Cancer Res. 14, 295. den Tonkelaar, E. M. en Kroes, R. (1971). Onderzoek naar de subacute hepatotoxieiteit van tetrachloorkoolstof en dimethylnitrosamine met behulp van leverfunctietesten. Versl. Meded. Volksgezondh. 33, 231. Druckrey, H., Preussmann, R., Ivankovic, S. u. Schm~ihl, D, (1967). Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen an BD-Ratten. Z. Krebsforsch. 69, 103. Druckrey, H., Steinhoff, D., Beuthner, H., Schneider, H. u. Kl~irner, P. (1963). Priifung yon Nitrit auf chronisch toxische Wirkung an Ratten. ArzneimitteI-Forsch. 13, 320. Ender, F., Havre, G., Heigebostad, A., Koppang, N., Madsen, R. & Ceh, L. (1964). Isolation and identification of a hepatotoxic factor in herring meal produced from sodium nitrite preserved herring. Naturwissenschaften 51,637. Magee, P. N. (1971). Toxicity of nitrosamines: Their possible human health hazards. Fd Cosmet. Toxicol. 9, 207. Magee, P. N. & Barnes, J. M. (1956). The production of malignant primary hepatic tumours in the rat by feeding dimethylnitrosamine. Br. J. Cancer 10, 114. Magee, P. N. & Barnes, J. M. (1962). Induction of kidney tumours in the rat with dimethylnitrosamine (N-nitrosodimethylamine). J. Path. Bact. 84, 19. Magee, P. N. & Barnes, J. M. (1967). Carcinogenic nitroso compounds. Adv. Cancer Res. 10, 163. Magee, P. N. & Schoental, R. (1964). Carcinogenesis by nitroso compounds. Br. reed. J. 20, 102. Musil, J. (1966). Der Einfluss einer chronischen Natriumnitrit-lntoxikation auf R.atten. Acta biol. reed. germ. 16, 388.

488

M.J. VAN LOGTEN, E. M. DEN TONKELAAR, R. KROES eta].

Sander, J. (1971). Untersuchungen tiber die Entstehung cancerogener Nitrosoverbindungen im Magen von Versuchstieren und ihre Bedeutung ftir den Menschen. Arzneimittel-Forsch. 21, 1572, 1707 & 2034. Sander, J. u. Biirkle, G. (1969). Induktion maligner Tumoren bei Ratten durch gleichzeitige Verf~itterung yon Nitrit und s e k u n d ~ e n Aminen. Z. Krebsforsch. 73, 54. Sander, J., Schweinsberg, F. u. Menz, H. P. (1968). Untersuchungen fiber die Entstehung cancerogener Nitrosaminen im Magen. Hoppe-Seyler's Z. physiol. Chem. 349, 1691. Schm~.hl, D. & Osswald, H. (1967). Carcinogenesis in different animal species by diethylnitrosamine. Experientia 23, 497.

Essai i long terme sur des rats de viande en boites trait6e au nitrite de sodium et au glucono-~-Iactone R~um6----L'article d~crit une ~tude de toxicit6 ~ long terme, o~ des rats ont consomm6, :~ raison de 4 0 ~ de leur r6gime, de la viande de conserve trait6e soit avec 0,5, soit avec 0,02% de nitrite de sodium avec ou sans glucono-,~-lactone. L'attention s'est port6e tout particuli6rement sur la d6tection d'6ventuels effets carcinog6nes. L'exp6rience 6tait con~:ue pour 6tudier la formation possible de nitrosamines b. partir du nitrite et des amines secondaires pr~sentes dans la viande, darts des conditions de pH acide et de pasteurisation. Parmi les param6tres 6tudi&s figurent les donn6es h6matologiques, clinico-biochimiques et histopathoIogiques, la pr6sence d'a-foetoprot6ine dans le s6rum et la teneur en A D N des noyaux de cellules h6patiques. On n'a relev6 aucun indice de modification pr6-n6oplastique ou de formation de tumeur que l'on aurait pu imputer b. la consommation de la viande de conserve en cause.

Langzeitiger Rattenversuch mit Dosenfleisch, behandelt mit Natriumnitrit und Glucono-~-lactone Zusammenfassung--Ein langzeitiger Toxizit/itsversuch wurde mit Ratten durchgeftihrt, an die Futter mit einem Gehalt yon 40% Konservenfleisch verabreicht wurde, das entweder mit 0,5 oder mit 0,02~0 Natriumnitrit mit oder ohne Glucono-3-1actone behandelt worden war. Dieser Versuch, bei dem der Feststellung einer m6glichen carcinogenen Wirkung besondere Aufmerksamkeit geschenkt wurde, war dazu bestimmt, die mtigliche Entstehung von Nitrosaminen aus Nitrit und den sekundAren Aminen im Fleisch unter den Bedingungen eines sauren pH-Werts und der Pasteurisierung zu untersuchen. Zu den beriicksichtigten Parametern geh6rten Hiimatologie, klinische Biochemie, Histopathologie, a-Foetoprotein im Serum und der DNS-Gehalt der Leberzellenkerne. Es gab kein Anzeichen einer pr~ineoplastischen Veriinderung oder Tumorentstehung, das der VerfiJtterung dieses Konservenfleischs zugeschrieben werden konnte.