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Groups of 48 male and 48 female mice were fed quillaia extract in the diet at levels of 0 (control), 0·1, 0·5 or 1·5% for 84 wk. The material had no adverse effect on the death rate or the incidence of histopathological findings, including tumours. However, there was a lower rate of body-weight gain at the 1·5% dietary level, and there were isolated statistically significant differences between the treated and control animals, mainly at the 1·5% dietary level, in the haematological examinations and in some absolute and relative organ weights of both sexes. It is concluded that, in mice, quillaia extract fed at levels up to 1·5% in the diet (approximately 2·2g/kg/day) did not exert a carcinogenic effect. The no-untoward-effect level from this study is considered to be 0·5% in the diet, giving an intake of approximately 0·7 g quillaia extract/kg/day.

Groups of 15 male and 15 female rats were given daily oral doses of 0 (control), 35 or 100 mg allyl caproate/kg/day, as a solution in oil, for 14 wk, and ten males and ten females were given 12 mg/kg/day for 13wk. There were no differences between treated and control rats in body weight, water intake, haematological examinations, analysis of urine, renal concentration tests or serum chemistry. There were slight increases in food intake at the highest dose level. The weight of the liver was increased with doses of 35 and 100 mg/kg/day, and all treated groups showed evidence of periportal vacuolation, which was dose-related in both incidence and severity and which, in the rats given 100 mg/kg/day, was accompanied by enlargement of the hepatocytes, focal periportal necrosis and bile-duct proliferation. Weights of the kidneys, spleen, stomach and small intestine were increased at the highest level of treatment. The small-intestine weight was also increased in females given 35 mg/ kg/day. On the basis of these findings it was not possible to establish a no-untoward-effect level for allyl caproate.

n-Amyl alcohol, dissolved in corn oil, was administered to rats by oral intubation at dose levels of 0 (control), 50, 150 or 1000 mg/kg body weight/day for 13 wk. The treatment had no demonstrable effect on body-weight gain, food and water consumption, haematological values, serum and urine analyses, renal function, organ weights or histopathology. The no-untoward-effect level was 1000 mg/kg/day. which is about 2000 times the estimated maximum likely intake by man.

Groups of 30 male and 30 female mice were given diets containing 0·1, 0·25, 0·5 or 1·0% Black PN for 80 wk, with groups of 60 mice of each sex as controls. There were no dose-related effects on body-weight gain, haematology or organ weights. The incidence of histopathological findings, including tumours, was not altered by the feeding of Black PN. It is concluded that the addition of Black PN to the diet of mice at levels up to 1·0% (equivalent to approximately 1300 mg/kg/day) for 80 wk does not elicit any long-term toxic or carcinogenic effects.

Erythrosine BS was given to groups of three male and three female pigs by mouth for 14 wk in doses of 0 (control), 167, 500 or 1500 mg/kg body weight/day. The treatment had no significant effects on body-weight gain, haematological findings, urine analyses or histopathology. Increases in the weights of the thyroid glands and decreases in serum levels of thyroxine were found at all treatment levels. It was therefore impossible to derive a no-untoward-effect level from this study.

Groups of 30 male and 30 female mice were fed diets containing 0·2, 0·4, 0·8 or 1·6% indigo carmine for 80 wk. A group of 60 male and 60 female mice served as controls. The treatment had no effects on the death rate, body-weight gain, organ weights or the results of the histopathological examination, including the incidence of tumours. There was a slight anaemia in mice given diets containing 0·8 or 1·6% indigo carmine. It is concluded that the feeding of indigo carmine to mice at dietary levels of up to 1·6% did not exert any carcinogenic effect. The no-untoward-effect level in this study was 0·4% of the diet.

Groups of 15 male and 15 female rats were given diet containing 0 (control), 100, 500 or 2500 ppm zinc dibutyldithiocarbamate (ZnDBDTC) for 17 wk. At the highest treatment level, the rate of body-weight gain of the females and the food intake of both sexes were reduced. There were no effects attributable to ZnDBDTC in the results of the haematological examination or the analyses of serum and urine. The relative weights of the liver and kidneys were increased in rats of both sexes given 2500 ppm ZnDBDTC. There were no differences between treated and control rats in the histological appearance of any of the tissues examined. The no-untoward-effect level was 500 ppm in the diet, providing an intake between 41 and 47 mg ZnDBDTC/kg/day.

Carmoisine was fed to mice at dietary levels of 0 (control), 0·01, 0·05, 0·25 or 1·25% for 80 wk. There were no adverse effects on mortality, weight gain, organ weights or the incidence of histopathological findings, including tumours. There was a mild anaemia in the mice given 1·25% carmoisine. It is concluded that carmoisine has no carcinogenic potential in mice at dietary levels up to 1·25% and that the no-untoward-effect level is 0·25% (equivalent to an intake of approximately 350 mg/kg/day).

Groups of 15 rats of each sex were given daily doses of 0, 10, 100 or 1000 mg isobutyl isobutyrate/kg by stomach tube for 18wk. The treatment had no significant effects on body-weight gain, food and water intake, haematological findings, serum and urine analyses, organ weights or histopathology. The no-untoward-effect level in this study was at least 1000mg/kg/day.

Groups of 15 rats of each sex were given diet containing 4, 8 or 16% caramel produced by either an ‘open-pan’ or ‘closed-pan’ ammonia process. A group of 25 rats of each sex served as controls. There was a reduced rate of body-weight gain at all dietary levels, although in the rats given 4% caramel there was evidence that this could be attributed to dilution of the diet. In the case of the open-pan material this also applied to the rats given 8% in the diet. There were reduced haemoglobin concentrations at the highest dietary level of both caramels, although similar findings at the lower levels were inconsistent and of doubtful significance. Lymphocytopenia was present at all dietary levels and was associated with lower weights of thymus and spleen at the two higher doses. The caecum weights were higher in the treated animals than in the controls, but this was not thought to represent a toxic effect. Increased relative liver and kidney weights suggested an effect on these organs. Changes in the weights of other organs were considered to be related to differences in body weight. A no-untoward-effect level could not be established for either of the caramels.

Groups of 30 male and 30 female mice were given diets containing 0·2, 0·4, 0·8 or 1·6% Sunset Yellow FCF for 80 wk. A group of 60 male and 60 female mice served as controls. The feeding of Sunset Yellow FCF did not adversely affect the death rate within the groups, the rate of body-weight gain, the organ weights or the haematological findings. The incidence and severity of the histopathological findings were similar in treated and control mice and there was no evidence of an increased incidence of tumours in the mice given Sunset Yellow FCF. It is concluded that in mice Sunset Yellow FCF fed at levels of up to 1·6% in the diet is not carcinogenic and does not exert any long-term toxic effects.

Groups of 15 male and 15 female rats were given diets containing 0 (control), 600, 2000 or 6000 ppm cyclohexylamine hydrochloride (CHA) for 13 wk. No changes in the haematological examinations, serum analyses or urinary cell excretion values were associated with CHA treatment. At the two higher dosage levels there was a reduced rate of body-weight gain and food intake. Paired feeding showed that the reduced food intake did not fully account for the failure to gain weight. The absorption of nutrients was normal but measurements of oxygen consumption suggested that an increased basal metabolic rate might account, in part, for the failure to gain weight. The renal concentrating ability was slightly reduced in the females given the highest dietary concentration of CHA. The weights of most of the organs were lower in the rats given diets containing 2000 or 6000 pp, CHA but this was related to the reduced body weight. The testis weight and relative testis weight were reduced and histopathological examination showed reduced spermatogenesis. However, the rats remained fertile and there was no evidence of abnormalities in their off-spring. A part from the effects on the testis, no changes were seen in the histopathological examination. The no-untoward-effect level established in this study was 600 ppm, a level equivalent to an intake of approximately 30 mg CHA/kg/day.

Rats were fed sorbitan monolaurate (Span 20) at dietary levels of 0 (control). 2·5, 5 or 10%, or lauric acid at 5%. for 13 wk. Dose-related reductions in the rate of body-weight gain were associated with reduced intakes of diet containing Span 20. There were similar reductions with lauric acid. Animals fed 5 or 10% Span 20 had reduced haemoglobin concentrations and packed cell volumes, but the other haematological parameters and serum-enzyme levels were normal. Variations in organ weight were mainly associated with the lower body weights. Those thought to be related to treatment were increases in relative liver and small-intestine weights in the rats given 10% and 5 or 10% diets, respectively, and increases in kidney weight at all dose levels. Periportal fat-containing vacuoles were demonstrated in the livers of the rats fed 10% Span 20 but the increased relative kidney weights were not accompanied by histological changes or impairment of renal function and no abnormal constituents were present in the urine. In view of the kidney enlargement it was not possible to estimate a no-untoward-effect level in this study.

Groups of 15 male and 15 female rats were given diet containing 0 (control), 0·2, 1·0 or 2·0% di-(2-ethylhexyl) phthalate (DEHP) for 17 wk. At the two higher treatment levels there was a reduced rate of body-weight gain and food intake. A paired-feeding study showed that the reduced food intake did not account fully for the reduced growth rate. There was a decreased packed cell volume in both sexes given 1 or 2% DEHP, and in the male rats given these levels the haemoglobin concentration was also reduced. However, there was no decrease in the total erythrocyte count. Renal concentrating and diluting ability was reduced in the females receiving 2% DEHP. There were no differences between treated and control rats in the results of the serum analyses. With the exception of the liver weights, which were higher, the absolute weights of most organs in the rats given 1 or 2% DEHP were lower than those of the controls, while the relative values were greater. This pattern was considered to be related to the lower body weights at autopsy. At these treatment levels, the relative testis weight was markedly decreased and histopathological examination revealed severe seminiferous tubular atrophy and cessation of spermatogenesis. The testis weight of the rats fed 0·2% DEHP was not reduced, but histologically there was evidence of decreased spermatogenesis. At wk 17, castration cells were observed in the pituitary at all treatment levels, the incidence being dose-related. There were no other histopathological changes attributable to DEHP treatment. In view of the testicular changes and the increase in liver weight at all treatment levels, it was not possible to establish a no-untoward-effect level for DEHP in this study.

Emulsifier YN was fed to groups of 50 male and 50 female mice at dietary levels of 0 (control), 2 or 6% for 80 wk. These treatments had no adverse effect on mortality, haematology, organ weights, the incidence and severity of histopathological changes or the incidence of tumours. The no-untoward-effect level for YN in this study was thus 6%, equivalent to an intake by the mouse of approximately 8.5 g/kg/day.

Caramel produced by a straight ammonia-catalysed ‘half open-half closed pan’ process was given at levels of 0 (control), 1, 3 or 6% in the diet to groups of 48 male and 48 female rats for 2 yr. In both sexes there was a reduced rate of body-weight gain at all dose levels. In the males, this was accompanied by a reduction in the cumulative food intake. There was evidence that dilution of the diet by the caramel also contributed to the reduction in body-weight gain. A reduction in numbers of white cells was associated in the early part of the study with a lymphocytopenia, which was present until wk 52 in the male rats fed the diet containing 6% caramel. The spleen weight was reduced in a dose-related manner. Changes in other organ weights were considered to be related to the difference in body weight between groups. A no-untoward-effect level was not established for the caramel used in this study, but there was no evidence of a carcinogenic effect.

Groups of 48 male and 50 female mice were fed diets containing 0 (control), 70, 700 or 3500 ppm Violet 6B for 80 wk. The faeces of all treated mice were violet-coloured but the urine appeared normal, suggesting that the dose was largely unabsorbed from the gastro-intestinal tract. The feeding of Violet 6B did not affect the mortality, the rate of body-weight gain or the haematological picture at 12, 28, 52 or 80 wk. The incidence of histopathological findings, including tumours, was not altered by the feeding of Violet 6B. It was concluded that the addition of Violet 6B to the diet of mice at levels up to 3500 ppm (equivalent to an intake of approximately 500 mg/kg/day) for 80 wk did not exert any carcinogenic effect.

Dietary administration of various chemically-unrelated hypolipidaemic ( viz. hypotriglyceridaemic) drugs can induce liver carcinomas in rats or mice. These drugs, in common with other compounds exhibiting hypolipidaemic activity (e.g. di-(2-ethylhexyl) phthalate), produce in rodents an initial hepatic response characterized by the presence of (1) liver enlargement unaccompanied by frank histological liver damage, (2) proliferation of the smooth endoplasmic reticulum and (3) a striking increase in peroxisome numbers. Hypolipidaemic drugs exert differential effects on the activities of hepatic peroxisomal enzymes. Massive increases in the activities of enzymes involved in the β-oxidation of fatty acids and of carnitine acetyltransferase overshadow the modest increases, if any, in the activities of catalase and certain oxidase enzymes. It is suggested that the bioavailability of catalase in the liver peroxisomes of rodents treated with hypolipidaemic drugs is insufficient to cope with the detoxication of injurious H 2 O 2 concentrations resulting from the considerably enhanced activity of H 2 O 2 -generating enzymes. Liver cells may thus be exposed to the cytotoxic or DNA-damaging potential of H 2 O 2 as a result of the breakdown in homeostasis, and such exposure could lead to the subsequent development of neoplasia in the rodent liver. Evidence is accumulating that in man (as well as in monkeys) hypolipidaemic drugs do not elicit the peroxisome proliferative response that is associated with the disposal of hepatic lipids in rodents. Consequently it would not be justified to extrapolate the findings of rodent hepatocarcinogenesis to humans. Because of differences in response between the rodent liver and human liver towards the action of certain microsomal-enzyme inducers (phenobarbital, dieldrin, DDT, Ponceau MX and safrole), a similar conclusion has been reached—that the hepatocarcinogenicity of these compounds demonstrable in the rodent is not relevant to man. Moreover microsomal-enzyme inducers share remarkably close parallels with hypolipidaemic drugs in respect of the initial hepatic reaction, a lack of mutagenic potential/DNA interaction and the nature of the carcinogenic response. From the available data it is concluded that the hepatic peroxisome response is an important aetiological factor in liver cancer induced by hypolipidaemic drugs in rodents. The absence of a peroxisomal response in man (and subhuman primates) coupled with important differences in the way rats and humans handle lipoprotein remnants bound to liver cells strongly indicates that chronic administration of hypolipidaemic drugs is unlikely to present a liver-cancer risk in man.

Toxic renal damage was induced in male and female rats by up to three sc injections of p-aminophenol or mercuric chloride. The urinary excretion of creatinine, alkaline phosphatase, lactic dehydrogenase and leucine aminopeptidase was determined over the 24-hr period following treatment, and was compared with histochemically demonstrated activities of the three enzymes in tissue sections and with histological examination. Creatinine excretion was not significantly affected by the doses of nephrotoxins used. Of the three urinary enzymes studied, lactic dehydrogenase was found to give the greatest response to toxic injury of the proximal tubules relative to control values. Enzyme excretion and enzyme histochemistry were found to have a similar sensitivity and reproduceability as indices of toxic damage in the 24-hr period following a single dose of the nephrotoxic agents, and were more sensitive than histology. Repeated doses caused sustained renal injury as judged by histochemistry and histology, but urinary enzyme excretion rapidly returned to normal after the initial dose. Furthermore, the histochemical and histological progress of recovery from a single dose of mercuric chloride did not correlate with urinary enzyme excretion. It was concluded that measurements of urinary enzymes in the rat are of extremely limited value in experimental toxicology.

The 7-day LD50 values for Erythrosine BS were similar in both sexes of rats and mice at 6·7–7·4 g/kg after a single oral administration and 0·32–0·40 g/kg following an ip injection. Erythrosine BS was given to groups of 15 male and 15 female rats at dietary concentrations of 0 (control), 0·25, 0·5, 1·0 or 2·0% for 13 wk. There were no effects attributable to treatment on the rate of body-weight gain, food intake, results of haematological examinations, serum analyses or renal function tests. The main findings in the study were caecal distension at all treatment levels, an increase in thyroid weight and pigmentation of the kidney tubules. The pigmentation was found in both sexes at the highest level and in males given 0·5 or 1·0% Erythrosine BS. The no-untoward-effect level was 0·25% of the diet, equivalent to an intake of 160–170 mg/kg/day.

At some dose levels, coumarin and 2,4-xylidine produce liver enlargement in rats without histological evidence of hepatocellular damage. When administered by gavage, coumarin produced this effect at a dose level of 135 mg/kg/day. Biochemically, cytochrome P -450 and amidopyrine demethylase were depressed by coumarin but remained at control values inrrespective of the dose of 2,4-xylidine administered. Histochemical and ultrastructural investigations on the enlarged livers produced by both coumarin and 2,4-xylidine revealed that the lysosomes in most of the centrilobular hepatocytes were translocated from their normal peribiliary distribution and many were grossly enlarged. Recognizable cell constituents were present in many of these lysosomes (autophagic vacuoles). In addition there was a centrilobular depression of glucose-6-phosphatase and aniline hydroxylase. Both the liver size and the severity of these cytological changes were related to the dose administered. They indicated the presence of liver damage despite a normal histological appearance. At a daily dose of 405 mg/kg, coumarin caused histologically demonstrable vacuolar degeneration and fatty change as well as liver enlargement. Coumarin at dose levels of 15 and 45 mg/kg/day and 2,4-xylidine at 10 mg/kg/day did not produce liver enlargement. At these dose-levels, no differences from controls treated with arachis oil alone were detected biochemically, histochemically or ultrastructurally.

The oral LD 50 values for trans -2-hexenal were 780–1130 and 1550–1750 mg/kg in rats and mice respectively. Intraperitoneal LD 50 values were 100–200 mg/kg in both species. trans -2-Hexenal was fed to rats at dietary levels of 0 (control), 260, 640, 1600 or 4000 ppm for 13 wk. The group given 4000 ppm showed a slight (but not statistically significant) reduction in growth rate associated with a reduced intake of a diet shown to be less palatable than the control diet. The treatments had no effect on haematology or serum and urine analyses but, under conditions of dehydration, the relatively low specific gravity and large volume of urine passed by males of the 4000-ppm group suggested a minimal degree of renal dysfunction. This was not, however, supported by urine analyses or kidney weights and histology. The only consistent abnormality was an increase in the ovary weights of all treated female rats (unaccompanied by any change in ovarian histology). This effect was not dose-related and in further studies, in which female rats were fed trans -2-hexenal at 4000 ppm of the diet for 13 wk and female rabbits were given daily oral doses of 200 mg/kg for 13 wk, no effect on ovary weight was found. A mild anaemia, increased stomach weights and acute gastric ulceration were seen in the treated rabbits, but it was considered likely that these effects were due to the high local concentrations of the test compound resulting from oral intubation. The no-untoward-effect level for trans -2-hexenal in the rat study was 1600 ppm of the diet (approximately 80 mg/kg/day).

Yellow 2G was fed to rats at dietary levels of 0 (control), 100, 1000 or 10,000 ppm for 13 wk. The colouring was decolorized in the caecum, but the colourless product turned red on exposure to the air. No adverse effects were seen in the rate of body-weight gain, or in the results of haematological examinations, serum chemistry, renal cell excretion and urinary concentration tests. No gross or micropathological effects were detected which could be attributed to the colouring. There were isolated changes in the weights of kidney, small intestine, adrenal glands and testes in rats receiving 1000 or 10,000 ppm Yellow 2G but these were not considered to be related to treatment. The caeca were enlarged in rats fed a diet containing 10,000 ppm. The no-adverse-effect level suggested is 1000 ppm (or approximately 80 mg/kg/day), a level 2000 times the expected human intake.

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 10,000 ppm (approximately 500 mg/kg/day).

The acute oral and intraperitoneal LD 50 s of diacetyl in rats lie between 3·0 and3·4 g/kg and between 0·40 and 0·65 g/kg, respectively. Rats were given 0 (control), 10, 30, 90 or 540 mg diacetyl/kg/day by oral intubation for 90days. No adverse effects were found at the three lowest dosage levels. The highest dosage level caused a decrease in weight gain, an increase in water consumption, anaemia, an increased leucocyte count and an increase in the relative weights of the liver, kidneys and adrenal and pituitary glands. Stomach lesions seen at autopsy showed necrosis with infiltration by inflammatory cells. The no-effect level (90 mg/kg/day) is equivalent to about 500 times the estimated dailyintake by man (0·17 mg/kg).

Summary Amines derived from Brown FK and from its two myotoxic components, 2,4-diamino-5-( p -sulphophenylazo)toluene and 1,3-diamino-4-( p -sulphophenylazo)benzene, were injected intravenously into rats in single doses of 3·13-25 mg/kg. The mixture of amines from Brown FK was also injected into mice in the same range of doses. Cardiac and muscular lesions were produced by the amines in both species. These amines are thought to be the toxic metabolites of Brown FK produced by microbiological degradation in the intestine. The finding that orally administered Brown FK is myotoxic in rats but not in mice is probably due to differences in the intestinal flora in the two species.

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 thougt to be spontaneous. The histopathological changes found were related to the presence of a mild infection rather than to treatment with amylcinnamic aldehyde. 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.

The LD 50 of cis -3-hexen-1-ol in rats and mice was in the range of 7–10 g/kg by theoral route and 0·4–0·6 g/kg by the intraperitoneal route. In the short-term study, rats were given cis -3-hexen-1-ol in the drinking water at levels of 0 (control), 310, 1250 or 5000 ppm. No effects were seen at 310 and 1250 ppm, but in males of the 5000-ppm group the intake of drinking water was reduced compared with controls, the relative kidney weight was increased and the urine collected in the first 2 hr after a water load was more concentrated. In females, the only finding was a transitory anaemia at this level. The no-effect level was 1250 ppm in the drinking water, equivalent to an intake of 120–150 mg/kg/day, or 5000 times the estimated intake in man.

Methyl amyl ketone was given to rats, by oral intubation in oil solution, at dose levels of 0 (control), 20, 100 or 500 mg/kg/day for 13 wk. There were no statistically significant differences between treated and control rats in the rate of body-weight gain, food or water consumption, haematological findings, or the results of renal concentration tests. The histological appearance of the tissues was unaffected by treatment. Ketone bodies were found in the urine of a few male rats given 20 mg/kg methyl amyl ketone but this was thought to be due to excretion of the unchanged compound. At 500 mg/kg/day, the liver weight was increased in both sexes and the kidney weight in males only. Some slight increases were also seen at the 100 mg/kg/day level. The excretion of cells in the urine was increased at the 100 and 500 mg/kg/day levels. A mammary adenocarcinoma developed in a female rat given 20 mg/kg/day but this was not thought to be associated with the treatment. The no-untoward-effect level established in this study was 20 mg methyl amyl ketone/kg body weight/day, a level approximately 100 times the estimated maximum likely intake in man.

Groups of mice were fed on diets containing 0 (control), 300, 1000, 3000 or 10,000 ppm Chocolate Brown FB for 80 wk. There were no dose-related effects on body-weight gain, haematology or organ weights. There was no evidence of any increase in tumour production in mice given Chocolate Brown FB. Pigment was found in the Kupffer cells of the liver, the phagocytic cells of the lymph nodes and spleen, the alveolar macrophages of the lungs and the epithelial cells of the small intestine in mice fed on a diet containing 10,000 ppm Chocolate Brown FB. The no-untoward-effect level of Chocolate Brown FB established in this study was 3000 ppm (equivalent to an intake of approximately 430 mg/kg/day).

A study has been made of the physicochemical basis of the early local tissue reaction elicited by repeated subcutaneous injections of water-soluble food colourings in the rat. With the exception of Erythrosine BS, the colourings were all absorbed equally rapidly and uniformly, whether from undamaged or from grossly fibrosed subcutaneous tissue. Hence retention at the injection site could not account for the observed differences in local reaction elicited by the colourings investigated. The physicochemical properties found to play a significant part in determining the early local reaction were surface activity, lipid solubility and protein-binding ability, particularly when the latter was associated with alteration of protein structure. No evidence could be found of aggregation of the colouring or of its ability to interact with calf-thymus DNA in vitro . The course of erythrocyte haemolysis and disintegration of cholesterol-lecithin mixed micelles under the influence of increasing concentrations of the sodium salts of Patent Blue V and Blue VRS appeared identical with the corresponding effects brought about by the surfactants sodium lauryl sulphate and triton X-100. Other triphenylmethane colourings possessing considerable surface activity were Light Green SF Yellowish, Fast Green FCF and Brilliant Blue FCF. Most xanthene colourings tested possessed high solubility in tricaprylin and in cholesterol-lecithin mixed micelles. While not displaying surface activity, these colourings affected the integrity of lipid membranes at very low concentrations, in a manner closely similar to that of the amphipathic compounds saponin and lysolecithin. The capacity to bind to protein and, in so doing, to alter protein structure resided in the colourings Light Green SF Yellowish, Fast Green FCF, Brilliant Blue FCF, Erythrosine BS and Indigo Carmine. None of the physicochemical properties described was present to any appreciable extent in the calcium salts of Patent Blue V or Blue VRS, in the xanthene Violamine R or in any azo colouring studied. The physical properties of a colouring were found to correlate closely with type of local reaction elicited by it. In the presence of a marked degree of surface activity, or of lipid solubility, extensive tissue necrosis resulted initially and determined the development of a type IV reaction. Binding with, and alteration of, protein modified the tissue changes by provoking a macrophage response and so leading to a type III reaction. Erythrosine BS was exceptional in that protein binding considerably diminished its lipophilic property, with consequent reduction in the degree of local damage. Colourings possessing none of the physical attributes described produced a mild effect (type I reaction) while a self-limiting reaction (type II) resulted if these properties were present only to a minimal degree. Our investigations lead to the conclusion that the type of the early local tissue reaction produced and, ultimately, the formation of sarcomas by some of the food colourings studied can be fully accounted for on the basis of the physicochemical characteristics of these compounds.

Brown FK was administered to rats at dietary levels of 0.0 (control), 0.001, 0.01, 0.1 and 1.0% for up to 21 wk and to miniature pigs in daily doses of 0, 100, 250 and 500 mg/kg for 24 wk. No effect was observed in either species with respect to growth rate, food consumption, organ weights or liver and kidney function. Apart from the observations described below, no histopathological changes were seen in any organs of the rats or pigs and no significant haematological abnormality was noted in either species. In rats, lipofuscin deposits were observed in the heart, skeletal muscle, kidney tubules, liver parenchymal cells and Kupffer cells at the 1.0% dietary level, in females at 13 wk and in both sexes at 21 wk. At this dietary level, one male showed myocardial changes similar in every respect to those seen following repeated daily administration of Brown FK by stomach tube and reported in paper III of this series (Grasso, Gaunt, Hall, Golberg & Batstone, Fd Cosmet. Toxicol. 1968, 6, 1). After 150 days' treatment, lipofuscin deposits were seen in renal tubular cells also at the 0.01 and 0.1% dietary levels. Deposition of lipofuscin was the principal change observed in pigs. It affected mainly the liver, in which it was observed in both males and females at all dosage levels of Brown FK and was accompanied by increased lysosomal enzyme activity, more marked at the higher dose levels. Lipofuscin was seen also in the heart in males, and here it was associated with an increased acid-phosphatase activity. It was not detectable in the heart in females. Lipofuscin was also present in the kidneys of both sexes, at the highest dose level in females and at all levels in males.

Groups of three Large White pigs of each sex were fed 0 (control), 100, 300 or 900mg Ponceau 4R/kg body weight/day for 90 days. One female from the highest dose level died on day 23, but the death was attributed to an enteritic infection and not to Ponceau 411 treatment. No differences between treated and control pigs were detected in growth, composition ofurine and serum, organ weights or histopathology. There was a slight reduction in the number of erythrocytes at wk 6 in males fed 900 mg/kg/day. The no-effect level found in this study for a period of 90 days was therefore 300 mg/kg/day.

The acute LD50s of p-hydroxybenzyl acetone were 1·3 and 1·4 g/kg when given orally and 0·70 and 0·35 g/kg when given intraperitoneally to male and female rats respectively. Rats were fed on diets containing 0·0 (control), 0·1, 0·2, 0·4 or 1·0% p-hydroxybenzyl acetone for 13 wk. No effects on body weight were recorded in animals on diets containing 0·4% or less of the flavouring and food and water consumption was similar in all test and control animals. Transient changes in haemoglobin and leucocyte levels at wk 7 in animals on the highest dosage were considered unlikely to be of toxicological significance. Serum and urine analyses were comparable in all groups, except that a positive reaction for acetone was found in the urine of all treated animals. Further tests showed, however, that this was related to the excretion of p-hydroxybenzyl acetone or its metabolites and not to any pathological change. No other effects were seen at the two lower levels but animals given the two higher dosage levels showed slight increases in liver, kidney and adrenal weight relative to body weight. The types and incidence of histological findings were comparable, however, in the control and highest dosage groups. The no-effect level of 100 mg/kg/day is 200 times greater than the estimated intake in man of 0·42 mg/kg/day

The induction of sarcomas at sites of repeated subcutaneous injection in rats was investigated with regard to the influence of the surface activity of the solutions injected on the yield of tumours. The administration of 0·5 ml 3% or 1·0 ml 2% aqueous solutions of Patent Blue V, sodium salt, by subcutaneous injection three times weekly for 66 wk induced local sarcomas in 11 18 and 9 16 rats, respectively. At these two concentrations the surface tension of water was depressed by 50%, a figure considerably greater than the critical level required to induce red-cell lysis in the rat (25% depression of surface tension). Doses of 1·5 ml sodium Patent Blue V administered as a 1% solution (producing 16% depression of surface tension) under the same conditions of treatment failed to induce any sarcomas in 20 rats. With sodium Blue VRS, sarcomas developed at the site of repeated injection in 6 19 rats when the colouring was administered as a 2% solution (producing 38·5% depression of surface tension) in doses of 0·5 ml. Injection of 1·0 ml of a 1% solution (11·5% depression of surface tension) reduced the incidence of sarcomas to 8 60 rats. The distinctive lipophilic property of Blue VRS appears to be responsible for the induction of these tumours. When the injections of 0·5 ml 2% Blue VRS were distributed between four subcutaneous sites in rotation, no sarcomas were elicited at any of the sites. Finally, using Tween 80 as a model compound, repeated injection of 2 ml 6% aqueous solution (52% depression of surface tension) three times weekly for 40 wk induced local sarcomas in 11 17 rats. The potentialities of the calcium ion in determining sarcoma production are illustrated in the different yields of tumours obtained with sodium and calcium cyclamate solutions injected subcutaneously for 107 wk. Administration of the sodium salt (0·5 ml of a 15% solution) failed to elicit any tumours, whereas administration of 1·0 ml 5% or 0·5 ml 15% calcium cyclamate produced sarcomas in 4 10 and 14 24 rats, respectively. In accordance with the results of earlier studies, it was found that the tissue reaction developing locally after a short series of injections correlated closely with the outcome of long-term tests. A progressive reaction was elicited at the sites of repeated subcutaneous injection of each of the following materials: 1 ml 1% sodium Blue VRS (type IV), 0·5 ml 15% and 1·0 ml 5% calcium cyclamate (type IV), and 2 ml 6% Tween 80 (type III). A self-limiting reaction was induced by 0·5 ml 15% sodium cyclamate (type I), and by 0·5 ml 2% sodium Blue VRS when the injections were distributed between four sites in rotation. Thus, within the range of compounds studied, the induction of local tissue changes and of sarcomas in rats was varied by modifying the surface activity of the solution injected or by introducing high concentrations of calcium ion. These observations suggest that such local sarcomas in the rat, produced by long-continued repeated injections into the same subcutaneous site, do not constitute a valid index of chemical carcinogenicity for purposes of safety evaluation.

Four samples of PVC containing 0, 0·17, 0·5 and 1·4% dibutyltin diacetate but otherwise identical in every respect, were used to evaluate two tissue culture methods frequently used in the safety testing of plastics. In one method (agar overlay technique), primary neonatal rat-kidney cells were grown in petri dishes, covered with 1% agar and stained with neutral red. Pieces of the plastics to be tested were then placed on the agar and the plates were examined 24 hr later. The area of neutral-red loss underneath each piece of PVC was taken as an index of toxicity. In the other method, primary neonatal rat-kidney cells were maintained in a growth medium made with serum previously used to extract dibutyltin diacetate from the plastics. In this case, lysosomal acid-phosphatase activity was used in addition to loss of neutral red to indicate cytotoxicity. With both methods, the extent of cell necrosis was in direct proportion to the concentration of dibutyltin diacetate in the plastics. The agar overlay method proved to be the more sensitive in detecting low concentrations of the cytotoxic agent in the plastics. The histochemical method demonstrated cytotoxic changes in the cells much earlier than did the loss of neutral red, but did not improve the sensitivity of the serum extract technique.

Sunset Yellow FCF was fed to pigs at levels of 0 (control), 250, 500 and 1000 mg/kg/day for 98 days. No differences were detected between test and control animals in weight gain, haematological indices, composition of urine, organ weights or serum levels of transaminases and urea. No abnormalities were seen at autopsy or on microscopical examination of the tissues. The no-effect level of the dyestuff in pigs was therefore greater than 1000 mg/kg/day for this period of feeding.

Isooctyl isodecyl nylonate was not lethal when given to rats orally or intraperitoneallyor to mice intraperitoneally in a single dose of up to 10 g/kg. The oral LD50 in mice was 8–10 g/kg. Rats were fed dietary levels of 0 (control), 125, 500, 2000 or 8000 ppm for 105 days. Therewere no effects on growth, food consumption, haematology or serum chemistry and no histopathological changes were seen in the treated animals. Kidney weights were significantly elevated in males in the 2000 and 8000 ppm groups and this increase was associated with an increased urinary cell excretion. Liver weight was increased in animals of the 8000 ppm group. The association of this increase with increased urinary ascorbic-acid excretion and increased activity of liver-microsomal processing enzymes is described. Adrenal and ovary weights were increased in females fed the highest dietary level. The level of nylonate at which no untoward effect was detected was 500 ppm which isapproximately equivalent to an intake of 25 mg/kg/day.

Groups of 30 male and 30 female weanling rats were fed for 2 yr on diets containing 0 (control), 1000, 3000, 10,000 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).

Acute oral toxicity studies with Brown FK gave LD50 values greater than 2 g/kg and 8 g/kg in mice and rats respectively. The values for intraperitoneal injection were 1·5–2 g/kg in mice and 0·75–1·15 g/kg in rats. When repeated daily oral doses ranging from 0·1 to 2 g/kg were given to rats, a precipitate weight loss and a vacuolar myopathy of the heart and skeletal muscles accompanied by lipofuscin formation occurred with doses of 0·5 g/kg and over. At the highest dose employed (2 g/kg) there was also hepatic centrilobular necrosis and renal tubular degeneration. Pretreatment with antibiotics reduced the incidence of muscle lesions. Repeated intraperitoneal injection did not have any effect on the heart or skeletal muscle. Only two of the six components of Brown FK, compound I (2,4-diamino-5-(p-sulphophenylazo)toluene) and compound II (1,3-diamino-4-(p-sulphophenylazo)benzene), were found to produce muscle damage after repeated oral doses of 0·5 g/kg. Comparable doses of mixtures containing the other components as well as I or II were considerably less toxic. After daily oral doses of 1 g/kg, muscle lesions were found in rats, rabbits and guinea-pigs, but none were seen in mice or hamsters. The results suggest that the intestinal microflora is responsible for the breakdown of the components of Brown FK to toxic products. Variations between species and between animals of the same species may be due to qualitative and quantitative differences in the intestinal microbial population.

Acute toxicity studies in rats and mice and a short-term study in rats have been carried out on Sunset Yellow FCF. The acute intraperitoneal LD50 ranged from 3·8 to 5·5 g/kg in rats and mice. Orally, doses up to 10 g/kg in rats and 6 g/kg in mice were tolerated without ill effect. Feeding of Sunset Yellow FCF to rats at dietary levels of 0·0 (control), 0·5, 1·0, 2·0 or 3·0% for 90 days evoked no adverse effect on growth or food consumption but there was slight diarrhoea throughout the study at 3% and during the first few weeks at 2%. There was no departure from normality in the haematological investigations or in the terminal liver and kidney function tests. At autopsy, the caecum was enlarged at the 2 and 3% levels and the testes at the 3% level. There were no histological changes attributable to Sunset Yellow FCF. A no-effect level of 1% Sunset Yellow FCF was established in the diet for 90 days, a level equivalent to 500 mg/kg/day.

A study has been made of the local lesions produced in rats (and in one instance in mice) by a short series of subcutaneous injections of food colourings belonging to various chemical categories. The resulting pathological changes have been separated into four main categories. In the type I response (Patent Blue V and Amaranth in rats and Blue VRS in mice) no appreciable connective tissue reaction was produced, while in the type II response (Erythrosine and Green S in rats) the connective tissue reaction developed to a limited degree. The nature of these responses suggested an inability of the test material to produce injury in the normal subcutaneous tissue or to interfere with the process of connective tissue repair whenever the normal integument became necrotic. These responses were produced by colourings which failed to produce sarcoma when injected repeatedly at the same site for 10 months or longer. Type III response (Light Green SF Yellowish, Brilliant Blue FCF and Fast Green FCF in rats) consisted mainly of an intense macrophage response involving macrophage necrosis and considerable fibroblastic proliferation. This effect closely resembles the tissue reaction reported by other workers following repeated injections of high doses of iron-dextran and of carboxymethylcellulose. Type IV response (Blue VRS and Eosine G in rats) resulted in the development of thick collagenous tissue with foci of persistent fibroblastic reaction. This lesion was found to correspond with the previously reported early stages of the fibrous reaction around subcutaneous implants of films of solid material. Colourings that have been reported by previous authors to induce subcutaneous sarcoma at the site of repeated injection produced either a type III or a type IV response. Thus a close correlation has been found to exist between the category of initial tissue response and the long-term outcome of repeated injections, in terms of the development of subcutaneous sarcoma at the site of injection. These results indicate the possibility that sarcoma production by the colourings under consideration results from derangement of connective tissue repair rather than from induction by a process of chemical carcinogenesis.

Orange RN was fed to rats at dietary levels of 0 (control), 60, 600, 1200 or 6000 ppm for 15 wk. This treatment had no effect on the growth or food intake of the animals or on the results of serum and urine analyses, except that the urine of animals on all but the lowest level of treatment was orange in colour. At 6000 ppm there was marked Heinz-body production, methaemoglobinaemia, anaemia and reticulocytosis together with enlargement of the spleen and increased splenic iron. At 1200 ppm these effects were present but less severe and at 600 ppm they were of borderline significance. There was also an increased water intake and decreased renal concentrating ability in the rats given the highest dosage level. The no-untoward-effect level found was 60 ppm, approximately equivalent to an intake of 3 mg/kg/day.

Feeding of Carmoisine to pigs at dose levels of 0 (control), 250, 500 or 1000 mg/kg/day for 90 days had no adverse effect on growth, and no differences between treated and control groups were evident in the haematological examination, serum chemistry, urine analyses, organ weights or histological examination. The no-effect level of 1000 mg/kg/day established by this 90-day study indicates that the pig is less sensitive to the effects of Carmoisine than is the rat, in which species a no-effect level of 250 mg/kg/day was previously established. Relative kidney weights were elevated in female rats fed 500 mg/kg/day.

Groups of rats were given daily doses of 0 (control), 150, 500 or 1000 mg isoamyl alcohol/kg body weight for 17 wk. There were no effects associated with treatment in the results of the haematological examinations, serum analyses, urinary cell counts, renal concentration tests or organ weights. A slightly reduced rate of body-weight gain at the highest dose level was shown to be due to a reduced food intake. Two rats given 1000 mg/kg/day died, but histopathological examination showed that these deaths were due to dosing into the lungs and not to any toxic effects of isoamyl alcohol. A female rat given 500 mg/kg/day developed a lipoma, which was not considered to be due to treatment. The other histopathological changes seen were related to mild infections in the animals and not to isoamyl alcohol. The no-untoward-effect level in this study was 1000 mg/kg/day, a level estimated to be 350–400 times the maximum likely intake in man.

Tolualdehyde was given to rats by stomach tube in daily doses of 0 (control), 50, 250 or 500 mg/kg body weight for 13 wk. There were no adverse effects on body-weight gain, food and water consumption, haematological examinations, serum analyses, renal concentration tests, urinary cell excretion or histopathology. There was a decrease in the relative pituitary weight in female rats given 500 mg tolualdehyde/kg/day for 6 or 13 wk. The weights and relative weights of small intestine in all treated groups were lower than those of the controls but the effect was not dose-related. This effect was not reproduced in a second study with groups of 30 female rats given either 0 or 500 mg tolualdehyde/kg/day for 13 wk. It is suggested that the small intestine weights in the control animals were unusually high in the main study. The no-untoward-effect level found was 250 mg/kg/day, a level approximately 1250 times the estimated maximum likely intake in man.