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Short-term toxicity study of chocolate brown HT in pigs
Toxicology, 11 (1978) 189--192 © Elsevier/North-Holland Scientific Publishers Ltd.
S H O R T - T E R M TOXICITY S T U D Y O F C H O C O L A T E B R O W N HT IN PIGS*
ROBIN J. HENDY, KENNETH R. BIYI~ERWORTH, IAN F. GAUNT, JEAN HOOSON and PAUL GRASSO
The British Industrial Biological Research Association, Woodrnansterne Road, Carshalton, Surrey SM5 4DS (Great Britain) (Received July 4th, 1978)
(Accepted July 20th, 1978)
SUMMARY
Chocolate Brown HT was given in the diet to groups of 3 male and 3 female pigs at dose levels of 0 (control), 5, 20 or 100 mg/kg/day for 13 weeks. No adverse effects were seen on b o d y weight gain, urine composition or the incidence o f histopathological lesions. The haemoglobin levels in all 3 t r e a t m e n t groups of male pigs at week 13 were significantly below the control values, b u t this effect was not considered t o be attributable to t r e a t m e n t with Chocolate Brown HT. The n o - u n t o w a r d ~ f f e c t level in this s t u d y was at least 100 mg/kg/day.
INTRODUCTION
Chocolate Brown HT (C.I. (1971) No. 20285) is the disodium salt of 4,4'-[ (2,4-dihydroxy-5-(hydroxymethyl)-m-phenylene)bis(azo) ] di-1naphthalene-sulphonic acid. It is permitted for use in the UK under the Colouring Matter in F o o d Regulations [1] and its review by the EEC authorities is pending. The principal uses of Chocolate Brown HT are in flour and sugar confectionery, canned meat, ice cream and sauces. The metabolic and toxicological investigations on this colouring have been summarized b y Drake et al. [2]. These workers found no evidence o f carcinogenic effects in a long-term study in mice fed levels up to 700 mg/kg/ day. However, in view o f some dubious findings, t h e y placed the no* This is an abridged paper. Copies of the full paper are available from the Editor on request, which should be accompanied by $5.00, or equivalent, to cover reproduction and postage.
189
untoward-effect level at 140 mg/kg/day, b u t considered that the true value may be considerably in excess of this figure. The findings included a reduced b o d y weight gain, decreased packed cell volume and total l e u c o c y t e c o u n t as well as an increased incidence of leucocytic infiltration of the liver at the highest level of treatment. A long-term study in rats has been c o m p l e t e d and will be reported shortly [3]. The present report describes a short-term feeding s t u d y with Chocolate Brown HT in pigs, performed as part of BIBRA's safety-evaluation programme. MATERIALS AND METHODS
Materials The Chocolate Brown HT used in these studies was supplied through the F o o d Colours Committee of the Chemical Industries Association. It conformed to the same specification as that used by Drake et al. [2] and thus complied with that of the Joint F A O / W H O Expert C o m m i t t e e on F o o d Additives [4 ]. Animals and diet 3 litters, each of 4 male and 4 female 10 week-old pigs of the Large White strain were obtained from a minimal
190
of glucose, ketones, bile salts and blood and the constituents o f the sediment obtained on centrifugation. After exsanguination under barbiturate anaesthesia, an a u t o p s y o f each pig was carried o u t during which the brain, pituitary, t h y r o i d , heart, stomach, small intestine, liver, spleen, kidneys, adrenals, gonads and caecum were weighed. Samples of these and of all major tissues and organs, and any other tissue appearing abnormal at autopsy, were preserved in 10% buffered formalin and prepared for histological examination. Statistical calculations are based on a level of significance of at least P = 0.05. RESULTS
There were no abnormalities in the condition and appearance of the pigs t h r o u g h o u t the study, apart from a slight, self-limiting diarrhoea in 2 female pigs (1 given 5 mg/kg and 1 given 20 mg/kg) during week 11. Faeces from both male and female pigs on the highest t r e a t m e n t level (100 mg/kg) were very dark brown and occasionally maroon in colour. There were no doserelated adverse effects on the body-weight gain. In the male pigs, the haemoglobin concentrations were significantly less than the control values in all 3 groups of Chocolate Brown HT-treated animals at the termination of the study. No such effects were seen in the female animals or in the values for either sex at week 5. The total leucocyte c o u n t o f the females given 100 mg/kg for 5 weeks was significantly higher than t h a t of the control pigs, b u t comparison of the individual data with t h a t of the litter-mate controls showed t h a t this 16% increase was due to 1 animal. The only other significant difference in the leucocyte c o u n t was a higher value in the males given 5 mg/kg, which was found at the end of the study. Also, in the same group, the methaemoglobin concentration was higher than the controls. Analysis of the urine showed no dose-related differences between control and treated pigs. No abnormal constituents were detected and the number of cells in the urine was similar in all groups. Absolute organ weights were unaltered by treatment, but there were scattered statistically significant differences in the relative organ weights between the treated and the control groups. With the adrenal glands and liver these differences were found only in the male animals and were unrelated to dose level. At a u t o p s y 1 male pig fed 5 mg/kg of the colouring was found to have a swollen ureter. Neither the tissues from this animal nor from any of the others showed histopathological changes which were not present in the controls. DISCUSSION
The dark colour of the faeces indicated either that a proportion of the
191
colour remained unabosrbed or t h a t a coloured metabolite was similarly unabsorbed. The observations of a m a r o o n colour suggest t h a t it was a metabolite t h a t was present. At the end of the s t u d y the mean leucocyte count and the methaemoglobin level of the pigs given 5 mg/kg were increased. These high values were due to increased levels in 1 pig and at autopsy this animal was f o u n d to have a swollen ureter. Despite the absence of histological abnormalities in the urinary tract of this animal, it is considered t h a t at least the raised leucocyte count might be related to this change. It is unlikely that this effect is causally linked to the administration of the colour. The lower haemoglobin levels in all the male treated groups do n o t appear to be related to the administration of the colour. The degree of variation in the control values was low and probably contributed to the statistical significance. There was no evidence o f a dose relationship and the m a x i m u m difference was 8% of the control value. In addition, there was no comparable effect in the female pigs. When litter-mates were compared across the 4 groups there was no systematic elevation or depression of values. Female pigs showed no such effect, neither were the red cell counts depressed in either sex. The approximate mean total c o n s u m p t i o n of the colour at the t r e a t m e n t levels was 13.5, 53.5 and 274 g/pig in males and 13.3, 50.1 and 259 g/pig in females. The present study showed t h a t the daily ingestion of up to 100 mg of Chocolate Brown HT/kg for up to 90 days in pigs produced no detectable u n t o w a r d effect. This no-untoward-effect level is comparable with those of approx. 250--300 mg/kg/day f o u n d in short-term studies in rats by Chambers, Hunter and Stevenson [5] and Hail, Lee and Fairweather [6] and of at least 140 mg/kg/day in mice by Drake et al. [2]. On the basis of the present work an Acceptable Daily Intake for man, after application of the traditional 100-fold safety factor, would be 1 mg/kg or a b o u t 70 mg for an adult. REFERENCES
1 Colouring Matter in Food Regulations, 1973. Statutory Instrument 1973, No. 1340. 2 J.J-P. Drake, K.R. Butterworth, I.F. Gaunt and J. Hardy, Toxicology, 10 (1978) 17. 3 F.M.B. Carpanini, K.R. Butterworth, I.F. Gaunt, I.S. Kiss, P. Grasso and S.D. Gangolli, BIBRA Research Report No. 1911975. 4 Joint FAO/WHO Expert Committee on Food Additives, Eighth Report, Tech. Rep. Ser. Wld Hlth Org., 309 (1965). 5 P.L. Chambers, C.G. Hunter and D.E. Stevenson, Food Cosmet. Toxicol., 4 (1966) 151. 6 D.E. Hall, F.S. Lee and F.A. Fairweather, Food Cosmet. Toxicol., 4 (1966) 143.
192
S H O R T - T E R M TOXICITY S T U D Y O F C H O C O L A T E B R O W N HT IN PIGS*
ROBIN J. HENDY, KENNETH R. BIYI~ERWORTH, IAN F. GAUNT, JEAN HOOSON and PAUL GRASSO
The British Industrial Biological Research Association, Woodrnansterne Road, Carshalton, Surrey SM5 4DS (Great Britain) (Received July 4th, 1978)
(Accepted July 20th, 1978)
SUMMARY
Chocolate Brown HT was given in the diet to groups of 3 male and 3 female pigs at dose levels of 0 (control), 5, 20 or 100 mg/kg/day for 13 weeks. No adverse effects were seen on b o d y weight gain, urine composition or the incidence o f histopathological lesions. The haemoglobin levels in all 3 t r e a t m e n t groups of male pigs at week 13 were significantly below the control values, b u t this effect was not considered t o be attributable to t r e a t m e n t with Chocolate Brown HT. The n o - u n t o w a r d ~ f f e c t level in this s t u d y was at least 100 mg/kg/day.
INTRODUCTION
Chocolate Brown HT (C.I. (1971) No. 20285) is the disodium salt of 4,4'-[ (2,4-dihydroxy-5-(hydroxymethyl)-m-phenylene)bis(azo) ] di-1naphthalene-sulphonic acid. It is permitted for use in the UK under the Colouring Matter in F o o d Regulations [1] and its review by the EEC authorities is pending. The principal uses of Chocolate Brown HT are in flour and sugar confectionery, canned meat, ice cream and sauces. The metabolic and toxicological investigations on this colouring have been summarized b y Drake et al. [2]. These workers found no evidence o f carcinogenic effects in a long-term study in mice fed levels up to 700 mg/kg/ day. However, in view o f some dubious findings, t h e y placed the no* This is an abridged paper. Copies of the full paper are available from the Editor on request, which should be accompanied by $5.00, or equivalent, to cover reproduction and postage.
189
untoward-effect level at 140 mg/kg/day, b u t considered that the true value may be considerably in excess of this figure. The findings included a reduced b o d y weight gain, decreased packed cell volume and total l e u c o c y t e c o u n t as well as an increased incidence of leucocytic infiltration of the liver at the highest level of treatment. A long-term study in rats has been c o m p l e t e d and will be reported shortly [3]. The present report describes a short-term feeding s t u d y with Chocolate Brown HT in pigs, performed as part of BIBRA's safety-evaluation programme. MATERIALS AND METHODS
Materials The Chocolate Brown HT used in these studies was supplied through the F o o d Colours Committee of the Chemical Industries Association. It conformed to the same specification as that used by Drake et al. [2] and thus complied with that of the Joint F A O / W H O Expert C o m m i t t e e on F o o d Additives [4 ]. Animals and diet 3 litters, each of 4 male and 4 female 10 week-old pigs of the Large White strain were obtained from a minimal
190
of glucose, ketones, bile salts and blood and the constituents o f the sediment obtained on centrifugation. After exsanguination under barbiturate anaesthesia, an a u t o p s y o f each pig was carried o u t during which the brain, pituitary, t h y r o i d , heart, stomach, small intestine, liver, spleen, kidneys, adrenals, gonads and caecum were weighed. Samples of these and of all major tissues and organs, and any other tissue appearing abnormal at autopsy, were preserved in 10% buffered formalin and prepared for histological examination. Statistical calculations are based on a level of significance of at least P = 0.05. RESULTS
There were no abnormalities in the condition and appearance of the pigs t h r o u g h o u t the study, apart from a slight, self-limiting diarrhoea in 2 female pigs (1 given 5 mg/kg and 1 given 20 mg/kg) during week 11. Faeces from both male and female pigs on the highest t r e a t m e n t level (100 mg/kg) were very dark brown and occasionally maroon in colour. There were no doserelated adverse effects on the body-weight gain. In the male pigs, the haemoglobin concentrations were significantly less than the control values in all 3 groups of Chocolate Brown HT-treated animals at the termination of the study. No such effects were seen in the female animals or in the values for either sex at week 5. The total leucocyte c o u n t o f the females given 100 mg/kg for 5 weeks was significantly higher than t h a t of the control pigs, b u t comparison of the individual data with t h a t of the litter-mate controls showed t h a t this 16% increase was due to 1 animal. The only other significant difference in the leucocyte c o u n t was a higher value in the males given 5 mg/kg, which was found at the end of the study. Also, in the same group, the methaemoglobin concentration was higher than the controls. Analysis of the urine showed no dose-related differences between control and treated pigs. No abnormal constituents were detected and the number of cells in the urine was similar in all groups. Absolute organ weights were unaltered by treatment, but there were scattered statistically significant differences in the relative organ weights between the treated and the control groups. With the adrenal glands and liver these differences were found only in the male animals and were unrelated to dose level. At a u t o p s y 1 male pig fed 5 mg/kg of the colouring was found to have a swollen ureter. Neither the tissues from this animal nor from any of the others showed histopathological changes which were not present in the controls. DISCUSSION
The dark colour of the faeces indicated either that a proportion of the
191
colour remained unabosrbed or t h a t a coloured metabolite was similarly unabsorbed. The observations of a m a r o o n colour suggest t h a t it was a metabolite t h a t was present. At the end of the s t u d y the mean leucocyte count and the methaemoglobin level of the pigs given 5 mg/kg were increased. These high values were due to increased levels in 1 pig and at autopsy this animal was f o u n d to have a swollen ureter. Despite the absence of histological abnormalities in the urinary tract of this animal, it is considered t h a t at least the raised leucocyte count might be related to this change. It is unlikely that this effect is causally linked to the administration of the colour. The lower haemoglobin levels in all the male treated groups do n o t appear to be related to the administration of the colour. The degree of variation in the control values was low and probably contributed to the statistical significance. There was no evidence o f a dose relationship and the m a x i m u m difference was 8% of the control value. In addition, there was no comparable effect in the female pigs. When litter-mates were compared across the 4 groups there was no systematic elevation or depression of values. Female pigs showed no such effect, neither were the red cell counts depressed in either sex. The approximate mean total c o n s u m p t i o n of the colour at the t r e a t m e n t levels was 13.5, 53.5 and 274 g/pig in males and 13.3, 50.1 and 259 g/pig in females. The present study showed t h a t the daily ingestion of up to 100 mg of Chocolate Brown HT/kg for up to 90 days in pigs produced no detectable u n t o w a r d effect. This no-untoward-effect level is comparable with those of approx. 250--300 mg/kg/day f o u n d in short-term studies in rats by Chambers, Hunter and Stevenson [5] and Hail, Lee and Fairweather [6] and of at least 140 mg/kg/day in mice by Drake et al. [2]. On the basis of the present work an Acceptable Daily Intake for man, after application of the traditional 100-fold safety factor, would be 1 mg/kg or a b o u t 70 mg for an adult. REFERENCES
1 Colouring Matter in Food Regulations, 1973. Statutory Instrument 1973, No. 1340. 2 J.J-P. Drake, K.R. Butterworth, I.F. Gaunt and J. Hardy, Toxicology, 10 (1978) 17. 3 F.M.B. Carpanini, K.R. Butterworth, I.F. Gaunt, I.S. Kiss, P. Grasso and S.D. Gangolli, BIBRA Research Report No. 1911975. 4 Joint FAO/WHO Expert Committee on Food Additives, Eighth Report, Tech. Rep. Ser. Wld Hlth Org., 309 (1965). 5 P.L. Chambers, C.G. Hunter and D.E. Stevenson, Food Cosmet. Toxicol., 4 (1966) 151. 6 D.E. Hall, F.S. Lee and F.A. Fairweather, Food Cosmet. Toxicol., 4 (1966) 143.
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