Search

The absorption, metabolism and excretion of 14C-labelled Ponceau 4R has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of 0.5 or 50 mg/kg body weight substantially all of the dose was excreted in the urine and faeces within 72 hr, with the majority being accounted for in the faeces. In all three species, naphthionic acid was the major urinary metabolite, whereas in the faeces naphthionic acid, 7-hydroxy-8-aminonaphthalene-1,3-disulphonic acid and unchanged dye were found. Pretreating male rats with unlabelled Ponceau 4R in the diet (50 mg/kg/day) for 28 days prior to dosing with the 14C-labelled colouring had no effect on the route of excretion or the time taken to eliminate the majority of the label. Following a single dose of 14C-labelled colouring to previously untreated rats, mice and guinea-pigs or to rats pretreated as above, no marked accumulation of radioactivity in any tissue was found, although tissue levels of radioactivity at 72 hr after dosing were higher in the pretreated rats than in those that were not pretreated. Pregnant rats eliminated a single oral dose of 14C-labelled colouring at a similar rate to non-pregnant females; however, some retention of radioactivity in the foetuses was found. In studies of absorption from isolated loops of small intestine containing 50, 500 or 5000 ppm Ponceau 4R, no significant absorption was detected in rats, but some absorption was seen in mice at the lowest concentration, and in the guinea-pig at the two higher concentrations.

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 rats were given metatartaric acid in the drinking-water in concentrations of 0 (control), 0.1, 0.5 or 3.0% for 18 wk. No effects associated with treatment were seen in the results of the haematological examinations and serum analyses. The treated animals consumed less water and food than the controls, probably because of the unpalatability of the test material. Administration of the 3% solution was associated in males with a reduced growth rate, some impairment of urine-concentrating ability during prolonged water deprivation (also seen in males on 0.5%) and histopathological changes in the stomach indicative of an inflammatory response in the submucosal layer. Both sexes of the 3% group showed an increase in relative kidney weight, without accompanying histopathological change. The no-untoward-effect level in this study was 0.1% metatartaric acid in the drinking-water, equivalent to a mean daily intake of 80 mg/kg body weight in the males and 130 mg/kg in the females.

The absorption, metabolism, tissue distribution and excretion of 14C-labelled Brown HT has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of either 50 or 250 mg Brown HT/kg, substantially all of the dose was excreted in the urine and faeces within 72 hr, with the majority (more than 80%) being accounted for in the faeces. A significant difference in urinary excretion of radioactivity was seen between male and female rats, as well as clear species differences at the two dose levels used. In all species studied, naphthionic acid was the major urinary metabolite, whereas in the faeces naphthionic acid, trace quantities of unchanged dye and at least two unidentified metabolites were found. Pregnant rats eliminated a single oral dose of 14C-labelled colouring at a rate similar to that in non-pregnant females, but some retention of radioactivity was found in the foetuses. Radioactivity was present in all tissues of male rats 24 hr after an oral dose of 250 mg 14C-labelled Brown HT/kg, with the highest concentrations in the gastro-intestinal tract, kidney and lymph nodes. Clearance from the gastro-intestinal tract was more rapid than from other tissues, but by day 7, the concentration of radioactivity (less than 0.001% of the dose/g) was similar in all tissues except the kidney and mesenteric lymph nodes. Similar results were obtained with animals pretreated for 21 days with either unlabelled or 14C-labelled Brown HT (250 mg/kg/day) prior to a radioactive dose. For most tissues examined, the concentration of radioactivity was greater with pretreatment than without. These results suggest that despite the rapid reduction and elimination of the major part of an oral dose of Brown HT, some colouring and/or metabolites accumulate in most tissues of male rats during repeated daily administration, but that only in the kidney and mesenteric lymph nodes is the accumulation tissue-specific. The accumulated radioactivity is cleared rapidly from most tissues on cessation of treatment. No significant absorption of either Brown HT, metabolites or subsidiary dyes was detected using isolated loops of small intestine.

Brown HT was fed to rats of both sexes over three generations at dietary concentrations designed to provide daily intakes of 0, 50, 250 and 500 mg Brown HT/kg body weight/day. During the study a number of females died or failed to nurse their litters. This was so severe following the first mating of F1 adults that the animals were remated to provide the next generation. None of these effects were related to treatment. Body weight and food and water intakes were not adversely affected by treatment. No effects of treatment were seen on reproductive performance or foetal and pup development, apart from slight evidence of a treatment-related retarded ossification of the third sternebrae. Organ weights at autopsy showed two changes, one of which was increased kidney weights which, although not present in every generation, seemed to be related to treatment. The other, increased caecum weights, occurred in adult high-dose females of early generations, but not in males or later generations of the study. Apart from brown coloration of tissues, macroscopic and microscopic examination revealed no treatment-related changes. It was concluded that the no-untoward-effect level in the present study was 250 mg Brown HT/kg/day.

Groups of 48 male and 48 female mice were fed for 80 wk on diet containing either 1·2% sorbic acid or 1·2% sorbic acid deliberately adulterated with 1000 ppm of specially prepared parasorbic acid. There were no statistically significant differences between the two treatments in body-weight gain, haematological examinations, organ weights or the incidences of histopathological findings, including tumours. Mortality was slightly higher in females given diet containing parasorbic acid than in those given only sorbic acid, but this difference was not considered to be related to treatment. It is concluded that the inclusion of parasorbic acid at a level of 1000 ppm in sorbic acid did not lead to an increase in the toxic effects of the latter, nor did the results suggest a carcinogenic effect.

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.

Phenylpropan-1-ol was added to the diet of groups of 15 male and 15 female rats to provide intakes of 0 (control), 10, 40 or 160 mg/kg/day for 13 wk. No effects on body weight, food intake, water intake, haematology, semi-quantitative analysis of urine, renal concentration and dilution tests, serum chemistry or the histological examination could be attributed to treatment. Increased liver weights at the highest dose level in both sexes and increased kidney weights at the two highest dose levels in males were considered to be related to treatment, but in view of the lack of histological abnormalities this study did not demonstrate whether these were toxic or adaptive effects. An isolated renal mesenchymal tumour was found in the females fed the highest dose level. It is concluded that the no-effect level in this study was 10 mg/kg/day.

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.

Groups of 15 male and 15 female rats were fed diet containing 0 (control), 1, 5 or 10% carnauba wax or 10% cellulose powder for 13 wk and groups of five rats of each sex were given these treatments, except the 1% carnauba wax, for 2 or 6 wk. Rats given 10% carnauba wax or 10% cellulose consumed more food than the controls but showed no differences in body weight, an effect attributed to the dilution of the diet by non-nutrient test materials. The study showed no treatment-related differences in body weights, water intakes, haematological values, serum-enzyme activities, urinary concentration and 'dilution' tests, organ weights or histological findings. The no-untoward-effect level for carnauba wax in the diet was 10%, which represented a mean intake of approximately 8.8 and 10.2 g/kg body weight/day in males and females, respectively.

Cyclohexylamine hydrochloride has been given in the diet to mice and to Wistar and DA rats for 13 weeks, to provide a constant intake of 400 mg of the base/kg/day. Significantly decreased food intake and body weight gain were found in both strains of rats but not mice. The metabolism of [14C]cyclohexylamine was widely different in Wistar and DA rats and in rats and mice, and these differences were not altered appreciably by chronic intake for 13 weeks. The differences in metabolism resulted in marked and persistent differences in the concentrations of the hydroxylated metabolites in the plasma and testes of treated animals with Wistar much greater than DA much greater than mice. After 7 and 13 weeks testicular atrophy was demonstrated in both strains of rats given cyclohexylamine diet by a decrease in organ weight and by histological changes. DA rats appeared more sensitive to testicular toxicity from cyclohexylamine than Wistar rats, while mice showed no evidence of testicular damage. These data show that the development of testicular toxicity is not related to the extent of hydroxylation. The concentrations of cyclohexylamine in the plasma and testes of the treated animals were lower in mice than in either strain of rats despite a similar daily intake. This suggests that species differences in pharmacokinetics may contribute to the apparent difference in sensitivity to testicular toxicity.

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.

Isoamyl salicylate was given to groups of 15 male and 15 female rats at dietary concentrations of 0 (control), 50, 500 or 5000 ppm for 13 wk. There was a decrease in weight gain at the highest dietary level accompanied by a reduced food intake, but a paired-feeding study showed that this was due to the diet's unpalatability. The females given the highest dietary level drank more water than the controls and produced slightly greater volumes of more dilute urine. The relative kidney weight was increased in rats on the 500 and 5000 ppm levels without any histopathological changes. It is concluded that isoamyl salicylate was exerting a mildly nephrotoxic effect. The relative liver weight was increased at the highest level of feeding but there were no other effects attributable to treatment. The no-untoward-effect level from this study was 50 ppm of the diet, providing a mean intake of approximately 4·7 mg/kg/day.

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.

Groups of 48 male and 48 female rats were given diets containing 0 (control), 1·5 or 10% sorbic acid for 2 yr. There was no increase in the rate or mortality in the treated rats and no changes attributable to treatment in the haematological examinations, analyses of serum, studies of renal function or histopathological examination. The relative liver and kidney weights were increased in the rats given 10% sorbic acid. The thyroid weight was increased at the same level in males but this was not thought to be due to sorbic acid treatment. This study showed no carcinogenic effect of sorbic acid at dietary levels up to 10%. The no-effect level established by this study was 1·5% of the diet, with changes of doubtful significance at the higher level (10%).

Groups of 48 male and 48 female rats were given diet containing 0 (control), 0·02, 0·1 or 0·5% ethyl methylphenylglycidate (EMPG) for 2 yr. There were no treatment-related effects on mortality, haematology, renal function, serum chemistry or organ weights. General observations, tests of motor co-ordination and histological examination of nerve tissues provided no evidence of neuropathy. Female rats given the highest dietary level (0·5%) weighed less than the controls for most of the study. Histological change in the pancreas, adrenal glands, lymph nodes and liver showed some increase in incidence in treated animals but the lesions were of types encountered in ageing rats. There was an increased incidence of pituitary tumours in treated females and of testicular interstitial-cell tumours in the males, but consideration of their background incidence in untreated rats and the lack of any dose relationship in their occurrence in this study indicated that these findings were unlikely to be related to treatment. It is concluded, therefore, that this study did not demonstrate a carcinogenic effect in rats given dietary levels of up to 0·5% EMPG and that the no-untoward-effect level was 0·1% of the diet, providing an EMPG intake of approximately 35 mg/kg/day in males and 60 mg/kg/day in females.

Carmine was fed continuously to groups of 54 males and 54 females at dietary levels providing 50, 150 or 500 mg/kg body weight/day for up to 109 wk. As a control, groups of 90 males and 90 females were fed the basal diet for the same period. The rats were derived from parents fed the same dietary levels for 60 days before mating and throughout pregnancy and were thus potentially exposed in utero. There were no adverse effects upon survival, growth or intakes of food and water. No changes associated with treatment were found during the periodic measurement of haematology or renal function, or in the serum chemistry or organ weights at the end of the study. Tumour incidence was not affected, and variations in the distribution of the non-tumour pathology were not considered to be due to treatment. It was concluded that carmine administered to rats in utero and for up to 109 wk is not carcinogenic and that the no-untoward-effect level is 500 mg carmine/kg body weight/day.

Groups of rats were given daily oral doses of 0 (control), 15, 50 or 150 mg methylphenylcarbinyl acetate/kg body weight for 13 wk. There were no effects on the rate of body-weight gain, although the food and water intakes were increased in the male rats given 150 mg/kg. The relative liver and kidney weights were increased in male rats given 50 or 150 mg/kg/day. There was an increased excretion of cells in the urine of male rats given 150 mg/kg for 6 wk. No histopathological changes were seen that could be related to treatment with methylphenylcarbinyl acetate. It was concluded that the no-untoward-effect level for methylphenylcarbinyl acetate when given to rats for 13 wk was 15 mg/kg.

Groups of 48 male and 48 female rats were fed quillaia extract in the diet at levels of 0 (controls), 0.3, 1.0 and 3.0% for 2 yr. The material had no adverse effects on death rate, water consumption, serum chemistry or haematological parameters or on the incidence of histopathological findings, including tumours. In the males given the 3% dietary level, the death rate, total leucocyte count at wk 108, incidence of kidney lesions and weights of the kidneys, heart and thyroid were all below the control values. These differences were explicable, however, in terms of a lowered body weight consequent on a decreased food intake. It is concluded that, in rats, quillaia extract fed at levels up to 3.0% in the diet did not have any carcinogenic effect. The no-untoward-effect level established in this was 3.0% in the diet, approximately equivalent to an intake of 1.5 g/kg/day.

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.

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.

The absorption, tissue distribution and excretion of 14 C-labelled citral has been investigated in the rat and the mouse. In both species, orally administered citral was rapidly absorbed from the gastro-intestinal tract, resulting in a fairly uniform distribution of label throughout the body of the mouse by 12 hr. In both species, radioactivity was excreted rapidly, the major route being the urinary tract. A significant proportion of the radioactivity was converted to 14 CO 2 by the rat. Most of the administered radioactivity was excreted within 72 hr by the rat and within 120 hr by the mouse. There was no evidence for long-term storage of citral in the body, and it is suggested that any hazard associated with tissue accumulation after prolonged exposure will be minimal.

Groups of 48 male and 48 female mice were given diets containing 0 (control), 0.01, 0.1 or 0.5% Chocolate Brown HT for 80 weeks. There was a slightly reduced body weight gain and a lower heart weight in males given the highest dietary level. At the same level at week 77 the packed cell volume and total leucocyte count values in females were lower than those of the controls. However, the relationship of these findings to the treatment was questionable, as was the increased incidence of leucocytic infiltration of the liver in the female mice at the 0.5% level of treatment. A brown coloration of internal organs seen at the highest treatment level was due probably to the Chocolate Brown HT. The distribution of tumours was similar in all groups and it was concluded that there was no evidence of a carcinogenic effect with doses up to 700 mg/kg/day while the no-untoward-effect level was greater than 140 mg/kg/day.

Hydratropic aldehyde, dissolved in corn oil, was given to rats by oral intubation at dose levels of 0 (control), 10. 50 or 500 mg/kg body weight/day for 15 wk. Differences between treated and control groups in the mean rate of body-weight gain, in food consumption and in renal function tests were not statistically significant. There were statistically significant increases in water consumption at the highest dose level and small reductions in haemoglobin concentration in rats given 50 or 500 mg/kg/day. The liver weight was increased in animals receiving 500 mg/kg/day, without any histopathological change. Slight increases in relative liver weight were noted with 10 and 50 mg/kg/day, but these were not considered to be related to treatment. Increased weights at 500 mg/kg/day were seen in the kidney, stomach and pituitary gland in both sexes and. in females alone, in the heart and spleen. The histological appearance of all tissues was similar in the test and control groups. The no-untoward-effect level was 10 mg/kg/day, which is more than 100 times the estimated maximum intake by man.

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.

Acute intraperitoneal studies on Chocolate Brown FB indicate an LD50 of 200 mg/kg in mice and a value within the range 250–500 mg/kg in rats. Rats and mice tolerated daily oral doses of 1 and 2 g/kg respectively, for 3 wk without any toxic manifestations. In a 90-day feeding study in rats given dietary levels of 0·0 (control), 0·3, 1·0 and 3·0%, the only untoward effect observed was a slight but non-significant retardation of growth in males at the 3·0% level. No departure from normality was seen with respect to food consumption, haematology, liver and kidney function and organ weights. Pigment was found present in the Kupffer cells of the liver, at the 3% level, and in the alimentary canal, lymph nodes and renal tubules at the 1 and 3% levels. Otherwise, pathological changes were comparable in incidence and severity between test and control groups. A no-effect level has been established for Chocolate Brown FB at 0·3% of the diet of rats for 90 days or 150 mg/kg/day.

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).

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

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.