Short-term toxicity of amyl cinnamic aldehyde in rats.

-Amyl cinnamic aldehyde was fed to rats at dietary levels of 0 (control), 80, 400 or 4000 ppm for 14 wk. No differences from controls were seen in the rate of body-weight gain, the consumption of food and water, haematological measurements, serum analyses, urinary cell excretion or renal concentration tests. There were increases in the relative liver and kidney weights of rats fed the highest dietary level, but these were not associated with any histopathological changes. One female rat at each of the two lower dietary levels developed small mammary adenomas, which were thought to be spontaneous. The histopathological changes found were related to the presence of a mild infection rather than to treatment with amyl cirmamicaldehyde. The no-untoward-effect level in this study was 400 ppm (approximately 23 mg/kg/day in males and 36 mg/kg/day in females). This is 500 times the likely maximum intake in man. I N T R O D U C T I O N Amyl cinnamic aldehyde (C6Hs-CH-C(CH2(CH2)a -CH3) -CHO; ~-amylcinnamaldehyde; a-pentylcinnamaldehyde; 2-pentyl-3-phenyl prop-2-enal) is a pale-yellow liquid with a floral odour and is used as a flavouring constituent in most categories of food. There are, at present, no specific regulations governing the use of flavourings in the UK. The Food Standards Committee (1965) in its Report on Flavouring Agents suggested 17 flavourings which should not be used in foods, but amyl cinnamic aldehyde was not one of these materials. I t is permitted in the USA under See. 121.1164 of the Code of Federal Regulations and was classified "generally recognised as safe" (as F E M A no. 2061) by the Expert Panel of the Flavor and Essence Manufacturers ' Association. The Council of Europe (1970) suggested a limit of 1 ppm in food. There are no published data on the metabolism of amyl cinnamic aldehyde. I t has been shown that ethyl cinnamic alcohol is oxidized to the corresponding acid but the presence of the substituent on the a carbon prevents fl oxidation (Williams, 1959). I t seems likely that a similar metabolic route would operate for amyl cinnamic aldehyde. The oral LDso of amyl cinnamic aldehyde in the rat has been reported by Jenner, Hagan, Taylor, Cook & Fitzhugh (1964) to be 3.73 g/kg. No effect was detected when amyl cinnamic aldehyde was fed to rats for 90 days at 122 ppm in the diet, providing a mean daily intake of 6"5 mg/kg (Oser, Carson & Oser, 1965). The present paper describes a short-term feeding study of the flavouring in rats, carded out as part of the BIBRA safety evaluation programme. 725 726 F . M . B . CARPANINI, I. F. GAUNT, M. G. WRIGHT, P. GRASSO and s. D. GANGOLLI EXPERIMENTAL Materials. Amyl cinnamic aldehyde was supplied by Bush Boake Allen Ltd. (London), and complied with the following specification: Purity, min. 97 ~ ; specific gravity (25/25°C), 0"963-0.968; refractive index (20°C), 1.552-1.559; acid number, max 5"0. Animals and diet. Rats of the CFE strain, obtained from an SPF breeding colony, were given ground Spillers' Laboratory Small Animal Diet and water ad lib. They were housed, five per cage, in an animal room maintained at 21 41 °C with a relative humidity of 50--60 ~. Loss of amyl cinnamic aldehyde from animal diet. A diet containing 10,000 ppm amyl cinnamic aldehyde was prepared and a sample was placed in a sealed container. The remainder was exposed to the air in an animal room for 48 hr. Methanol extracts of these samples were analysed for amyl cinnamic aldehyde content using a Pye 104 dual-flame ionization gas chromatograph fitted with a 5 ft glass column packed with Celite 545 (100/120 mesh) impregnated with I 0 ~ polyethylene glycol adipate. The column temperature was 190°C. Only 1 ~ of the flavouring was lost from the diet exposed to animal-room conditions for 48 hr. As this was not considered to be a significant loss, amyl cinnamic aldehyde was administered in the food, which was prepared every 4-5 days. Experimental design and conduct Amyl cinnamic aldehyde was fed to groups of 15 male rats (body weight 100-125 g) and 15 females (body weight 90-120 g) for 14 wk at dietary levels of 0 (control), 80, 400 or 4000 ppm. Additional groups of five rats of each sex were given 0 (control), 400 or 4000 ppm amyl cinnamic aldehyde in the diet for 2 or 6 wk. The animals were weighed initially and then weekly up to wk 14. Food and water consumption were measured over the 24-hr period preceding each weighing. After the appropriate period of feeding the rats were killed by exsanguination under barbiturate anaesthesia. The blood so obtained was used for haematological studies and serum chemistry. The haematological investigations consisted of measurement of haemoglobin content, packed cell volume, and counts of erythrocytes, total leucocytes and individual types of leucocytes. Reticulocytes were counted in the samples from control rats and those fed the highest level of amyl cinnamic aldehyde (4000 ppm). Serum was analysed for the content of urea, glucose, total protein and albumin and for the activities of glutamie-oxalacetic and glutamic-pyruvie transaminases and of lactic dehydrogenase. A measurement of urinary concentrating ability was made during the final week of treatment by measuring the specific gravity and volume of urine produced in a 6-hr period of water deprivation. At the same time, samples of the urine were examined for appearance, microscopic constituents and content of cells, glucose, ketones, bile salts and blood. At wk 6 and 13, urine was also collected from the rats over a 2-hr period following a water load of 25 ml]kg and between 16 and 20 hr after the water load. Each animal was given an autopsy, during which any macroscopic abnormalities were noted and the brain, pituitary, thyroid, heart, liver, spleen, kidneys, adrenals, gonads, stomach, small intestine and caecum were weighed. Samples of these organs and of salivary gland, trachea, aorta, thymus, lymph nodes, urinary bladder, colon, rectum, pancreas, uterus, skeletal muscle and any other tissue that appeared to be abnormal were fixed in 10 buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin for microscopic examination. SHORT-TERM TOXICITY OF AMYL CINNAMIC ALDEHYDE 727 RESULTS One female rat fed diet containing 400 ppm amyl cinnamic aldehyde showed signs of respiratory distress and lost weight rapidly during wk 14. At autopsy consolidation of parts of the lungs was seen and histopathological examination of the tissues revealed pneumonia and an abscess of the renal pelvis. During the last 3 wk of the study, a subcutaneous mass (0-5-1.0 cm in diameter) was noticed in two female rats (one fed 80 ppm and the other 400 ppm amyl cinnamic aldehyde). At autopsy these masses were seen to be encapsulated and lying free in the subcutaneous space and to consist of firm, white lobular tissue. Histopathological examination showed both to be mammary adenomas. Apart from these three rats, no abnormalities were seen in the behaviour or appearance of the animals. There were no statistically significant differences between treated and control groups in rate of body-weight gain, food consumption or water consumption (Table 1). The mean intakes of amyl cinnamic aldehyde over the 14-wk period were 6"I, 29.9 and 287.3 mg/kg/day by the males of the groups given 80, 400 and 4000 ppm in the diet and 6.7, 34-9 and 320.3 mg/kg/day by the females. In addition, there were no significant differences between treated and control groups in the results of the haematological examinations (Table 2), serum analyses (Table 3) or urinary cell excretion and renal concentration tests (Table 4). The only statistically significant differences in the absolute organ weights of treated and control rats were seen after treatment for 6 wk. These consisted of lower stomach weights in the males given 400 ppm amyl cinnamic aldehyde and lower small-intestine weights in the females given 4000 ppm. In the latter case, the difference was not significant when the organ weights were expressed relative to body weight (Table 5). In addition, at the highest dietary level (4000 ppm), there were significant increases in relative liver weights in both sexes after 14 wk treatment and in males after 6 wk. Relative kidney weights were also increased after 14 wk treatment at 4000 ppm. At autopsy there were red, patchy lungs in three male rats (two at the 80 ppm level and one control) and pale kidneys in some male rats in all groups including controls. Histopathological examination revealed vacuolation of some liver cells, protein casts in the kidney tubules and signs of chronic lung infection. The incidence of these findings was low and was similar in both treated and control rats. DISCUSSION There were signs ofiU health in a female rat fed amyl cinnamic aldehyde at 400 ppm in the diet for 14 wk but the findings at autopsy and the histopathological examination of the tissues showed this to be due to chronic lung and kidney infection and therefore it could not be related to treatment. Small mammary adenomas were found in two treated female rats. Neither of these rats was from the group fed the highest level of amyl cinnamic aldehyde and tumours of this type are known to occur spontaneously in up to 65 ~o of older female rats (Gaunt, Brantom, Grasso, Creasey & Gangolli, 1972; Gaunt, Carpanini, Crrasso, Kiss & GangoUi, 1972; Gaunt, Carpanini, Grasso & Lansdown, 1972). Thus, the presence of these two benign tumours could not be related to treatment with amyl cinnamic aldehyde. Similarly, none of the other histopathological findings in this study could be associated with the intake of diets containing amyl cinnamic aldehyde. F.C.T. l l ] ~ " ! 728 F . M . B . CARPANINI, I. F. GAUNT, M. G. WRIGHT, P. GRASSO and s. D. GANGOLLI