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Short-term study of Chocolate Brown HT in rats
Fd Cosmet. Toxicol. Vol. 4, pp. 151-155. Pergamon Press 1966. Printed in Great Britain
Short-term Study of Chocolate Brown HT hi Rats P. L. CX-IAMBERS,C. G. HUNTER a n d D. E. STEVENSON
Tunstall Laboratory, Broad Oak Road, Sittingbourne, Kent, 'England (Received 9 June 1965) Abstract---ChocolateBrown HT was fed to rats at dietary levels of 0-0, 0.02, 0'06, 0-20, 0-60, 1.0 and 2 ~o for 90 days. No adverse effect was observed in respect of appearance, behaviour or survival of animals. Although body-weight gain in treated rats did not differ significantly from that of the controls, when adjustment was made for the total food intake, the slight growth retardation observed in males at the 1 and 2 ~o levels and in females at the highest level became significant. Haematological examination revealed slight but significant decreases in haemoglobhl, red cell count and haemotocrit in male rats on the highest dietary level. In the biochemical studies, reductions in the blood urea levels occurred in both sexes and were significant in all groups except at the 0-06 and 0'6~0 levels. The uneven group distribution of this effect suggests that a cause other than Chocolate Brown HT is likely. Increases in total serum protein were seen in males on the 0"6 and I ~o levels. The absolute weights of the heart, kidneys, liver, spleen and testes remained unaffected at all levels. There was no evidence of pathological damage at any dietary level of the colouring administered but pigment was seen at the two highest levels in certain intestinal cells, lymph nodes and cells of the proximal convoluted tubes of the kidney. On the basis of these results a maximum no-effect level of Chocolate Brown .HT has been established at 0"6 ~ in the diet of rats for 90 days. INTRODUCTION I n c o n j u n c t i o n w i t h t h e s h o r t - t e r m study o f C h o c o l a t e B r o w n H T in the r a t r e p o r t e d in the previous p a p e r (Hall, Lee & F a i r w e a t h e r , 1966) a similar study was u n d e r t a k e n with a view to c o m p a r i n g t h e results o b t a i n e d b y two i n d e p e n d e n t g r o u p s o f w o r k e r s e m p l o y i n g p r o c e d u r e s t h a t were essentially similar b u t differed in p o i n t s o f detail. T h e status o f C h o c o late B r o w n H T as a f o o d c o l o u r i n g in t h e U K has been outlined b y H a l l et al. (1966), w h o also n o t e d t h a t t h e r e were no p u b l i s h e d d a t a on the toxicity o f this colouring. EXPERIMENTAL
Materials. T h e s a m p l e o f C h o c o l a t e B r o w n H T used in t h e s h o r t - t e r m study in rats was supplied b y t h e same source a n d h a d t h e same chemical specification as described by H a l l et al. (1966). Animals. W e a n l i n g C a r w o r t h F a r m rats r e a r e d in t h e T u n s t a i l L a b o r a t o r y b r e e d i n g unit were used. Experimental design and conduct. G r o u p s o f 12 rats, equally divided b y sex a n d h o u s e d i n d i v i d u a l l y in cages, were m a i n t a i n e d on diets c o n t a i n i n g C h o c o l a t e B r o w n H T at levels o f 0.02, 0.06, 0-20, 0.60, 1.0 a n d 2.0 ~ for 90 days. A c o n t r o l g r o u p consisting o f 24 m a l e a n d 24 female rats received t h e basic diet for t h e s a m e p e r i o d . F o o d ( p o w d e r e d 86 diet) a n d Water were supplied ad lib. B o d y weight was r e c o r d e d weekly a n d f o o d c o n s u m p t i o n twice weekly. H a e m a t o l o g i c a l studies c o n d u c t e d t e r m i n a l l y involved m e a s u r e m e n t s o f h a e m o g l o b i n , t o t a l red a n d white cell counts a n d h a e m a t o c r i t . S e r u m levels o f p r o t e i n a n d u r e a were
151
152
P.L. CHAMBERS, C. G. HUNTER a n d D. E. STEVENSON
also determined. A t autopsy, the heart, liver, spleen, kidneys a n d testes were weighed a n d submitted to m a c r o p a t h o l o g i c a l e x a m i n a t i o n , t o g e t h e r with the o t h e r m a j o r organs. Paraffin sections o f the m a j o r organs, stained with h a e m a t o x y l i n a n d eosin, were exa m i n e d histologically. RESULTS A l t h o u g h some rats h a d m i n o r r e s p i r a t o r y d i s o r d e r s the r e m a i n d e r in all the g r o u p s r e m a i n e d healthy. Ten deaths were recorded, but these were evenly d i s t r i b u t e d t h r o u g h o u t the c o n t r o l and test groups. T h e rats grew well a n d the m e a n final b o d y weights showed no significant change (Table 1). However, t h e non-significant reductions in weight gain at t h e 1 a n d 2 % levels b e c a m e significant when account was t a k e n o f the fact t h a t the g r o u p s involved c o n s u m e d m o r e f o o d t h a n their respective controls a n d an a p p r o p r i a t e c o r r e c t i o n i n t r o d u c e d to m a k e allowance for the lowered efficiency o f f o o d utilization at the higher d i e t a r y levels (Table 1). H a e m a t o l o g i c a l e x a m i n a t i o n revealed slight, b u t nevertheless significant, reductions in h a e m o g l o b i n , red cell c o u n t a n d p a c k e d cell v o l u m e in males on the 2 % level (Table 2). In the biochemical studies, t o t a l serum p r o t e i n was increased in m a l e rats on the 0.6 a n d 1.0 % levels b u t n o t at 2 ~o o r any o t h e r level. F e m a l e g r o u p s r e m a i n e d unaffected (Table 3). The b l o o d u r e a c o n c e n t r a t i o n was depressed in each test g r o u p o f b o t h sexes a n d with the exception o f rats on 0.06 a n d 0.6 % o f the c o l o u r i n g the reductions were significant (Table 3). There were no i m p o r t a n t changes at any level in the a b s o l u t e weights o f the heart, kidneys, liver, spleen and testes (Table I). Table I. E~rectof 90-day feeding of 0-2 % Chocolate Brown HT on growth, food consumption and organ weights of rats Mean group Dietary terminal body weight level (~0) Uncorrected Corrected'l-
Total food intake (g)
Heart
Liver
Spleen
Kidneys
Testes
13.26 14.13 12-27 12-63 13.38 12.29 11-72
0-84 0.79 0.75 i.005 0.78 0.73 0-95
2-17 2-28 2-14 2-08 2.35 2.24 2.14
3"32 3"18 3"23 3'41 3"22 3"26 3"50
8"32 7-98 8-42 7"85 8'02 8"26 7-82
0"62 0'62 0"61 0-60 0.60 0"69 0"65
1"44 1"38 1-45 1"34 1"41 1"47 1-39
Absolute organ weight (g)
0.0 0.02 0.06 0.2 0.6 1.0 2.0
343 357 332 333 337 326 321
344 350 344 341 337 324* 303***
1962 2017 1903 1924 1970 1984 2082
Males 1-09 1-11 1.05 1.04 1-09 1.09 1.07
0"0 0"02 0'06 0"2 0"6 1"0 2-0
224 217 226 217 222 226 211
223 225 226 223 223 218 204**
1636 1505 1630 1527 1612 1752 1736
0"81 0-81 0"82 0'77 0"76 0'85 0"76
Females
Values marked with asterisks differ significantly from those of controls: *P<0"05; **P
S H O R T - T E R M TOXICITY OF C H O C O L A T E B R O W N H T
8
&
8
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V
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'~ I ~
e,i F',
±~,-=
8
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153
154
P.L. CHAMBERS,C. G. HUNTER and D. E. STEVENSON Table 3. Blood urea and serum protein levels in rats fed Chocolate Brown H T at dietary levels o! O--2% for 90 days
Dietary level (%)
Blood urea (mg/100ml)
0'0 0.02 0"06 0'2 0"6 1"0 2'0
Males 42.65 37.18"* 39-25 36"40*** 39"36 35"18"#* 35"30***
0'0 0'02 0"06 0"2 0"6 1"0 2'0
42'30 38"91" 40"18 35"54*** 41"00 35-00*** 36'80
Total serum p r o t e i n (g/100ml)
•
6'50 6"45 6"63 6"66 6"72* 6"74* 6"49
Females
6-65 6"53 6"67 6'65 6.64 6"40 6.73
Values marked with asterisks differ significantly from those of controls: *P
SHORT-TERM TOXICITY OF CHOCOLATE BROWN I-IT
155
REFERENCES Hall, D. E., Lee, F. S. & Fairweather, F. A. (1966). Acute (mouse and rat) and short-term (rat) toxicity studies on Chocolate Brown HT. Fd Cosmet. Toxicol. 4, 143. E t u d e s ~ court terme de l'action du colorant Chocolat Brun H T c h e z le rat R~sum~---Des rats ont 6t(~ soumis ~. des r~gimes alimentaires contenant 0,0; 0,02; 0,06; 0,20; 0,60; 1 et 2 % de colorant Chocolat Brun HT durant une p6riode de 90 jours. Aucune action d~favorable n'a 6t6 o b s e r v ~ sur l'6tat g~n6ral, sur le comportement ou sur la survie de ces animaux. Bien que le gain pond6ral chez les rats soumis b. ces r6gimes n'ait pas diff6r6 d'une fad;on nette de celui des t6moins, cependant, s i r o n tient compte des ingesta totaux, le 16get retard de croissance constat6 chez les animaux des deux sexes recevant des rb~gimescontenant 1 et 2 % de colorant, s'est av6r6 significatif. Chez les rat miles soumis ~. des r(~gimes contenant le pourcentage le plus 61ev6 de colorant, des examens h6matologiques ont montr6 une baisse l~g~re mais nette du taux d'h~moglobine, du nombre des globules rouges et de l'h6matocrite. Les ~preuves biologiques ont montr6 une baisse de l'ur~e sanguine chez les animaux des deux sexes, marqu6e chez tousles groupes saul ceux soumis b. des r6gimes contenant 0,06 et 0,6 % de colorant; le caract~re irr6gulier de cette baisse parmi les divers groupes laisse penser qu'un facteur autre que le colorant Chocolat Brun HT a 6t6 en cause. Chez les rats mfiles soumis ~. des r~gimes contenant 0,6 et 1% de colorant, il a 6t6 constat6 une augmentation de la prot6inimie. Le poids absolu du coeur, des reins, du role, de la rate et des testicules n'a pas 6t6 affect6 darts tousles groupes. Aucun signe de 16sion pathologique n'a ~t~ constat6 chez les rats des diff6rents groupes recevant le colorant, si ce n'est la pr6sence de pigments au niveau de certaines cellules intestinales, des glandes lymphatiques et des cellules des tubes contourn~s du rein chez les animaux soumis aux r~gimes contenant les pourcentages les plus 61ev6s de colorant. Selon les r~sultats de ces 6tudes, il a 6t(~ 6tabli que 0,05 % de colorant Chocolat Brun HT administr(~ au rat durant 90 jours n'entraine aucune action d~favorable. K u r z z e i t i g e Untersuchung der Wirkung yon Schokoladenbraun H T a u f R a t t e n Zusammenfassung--Schokoladenbraun HT wurde 90 Tage lang an Ratten in Konzentrationen von 0,0; 0,02; 0,06; 0,20; 0,60; 1,0 und 2% im Futter verabreicht. Es wurde keine nachteilige Wirkung hinsichtlich des Aussehens, des Verhaltens und des ~berlebens der Tiere beobachtet. Wenn auch bei Anpassung der Gesamtfuttermenge die Zunahme des K6rpergewichts der behandelten Ratten sich nicht signifikant yon derjenigen der Kontrolltiere unterschied, so wurde doch eine leichte Wachstumsverz6gerung bei den m~nnlichen Tieren, die 1 und 2% erhielten, und bei weiblichen Tieren der h6chsten Konzentrationsgruppe signifikant. Die h~.matologische Untersuchung zeigte leichte, aber signifikante Verminderungen des H~.moglobins, der Erythrozytenzahl und des H§.matokrits bei m~.nnlichen Ratten mit der h6chsten Konzentration im Futter. Bei den biochemischen Untersuchungen wurden Verminderungen des Harnstoffgehalts des Blutes bei beiden Geschlechtern beobachtet, und sie waren in allen Gruppen ausser denen mit einer Konzentration yon 0,06 und 0,6 % im Futter signifikant. Die ungleichm~ssige Gruppenverteilung dieses Effekts l~.sst vermuten, dass eine andere Ursache als das Schokoladenbraun HI" dafi~r verantwortlich ist. Eine Zunahme des Gesamtserumproteins wurde bei m~.nnlichen Tieren beobachtet, die 0,6 und 1% erhielten. Die absoluten Gewichte yon Herz, Nieren, Leber, Milz und Hoden blieben bei allen Konzentrationen unbeeinflusst. Es gab keine Anzeichen pathologischer Sch~.den bei irgendeiner Konzentration des Farbstoffs, jedoch wurde bei den beiden h6chsten Konzentrationen Pigment in bestimmten intestinalen Zellen, Lymph Knoden und Zellen der proximalen gewundenen Harnkan~ilchen der Niere festgestellt. Auf der Grundlage dieser Ergebnisse wurde eine maximale wirkungslose Dosis von Schokoladenbraun H T mit 0,5 ~o im Futter von Ratten ftir die Dauer von 90 Tagen festgesteUt.
Short-term Study of Chocolate Brown HT hi Rats P. L. CX-IAMBERS,C. G. HUNTER a n d D. E. STEVENSON
Tunstall Laboratory, Broad Oak Road, Sittingbourne, Kent, 'England (Received 9 June 1965) Abstract---ChocolateBrown HT was fed to rats at dietary levels of 0-0, 0.02, 0'06, 0-20, 0-60, 1.0 and 2 ~o for 90 days. No adverse effect was observed in respect of appearance, behaviour or survival of animals. Although body-weight gain in treated rats did not differ significantly from that of the controls, when adjustment was made for the total food intake, the slight growth retardation observed in males at the 1 and 2 ~o levels and in females at the highest level became significant. Haematological examination revealed slight but significant decreases in haemoglobhl, red cell count and haemotocrit in male rats on the highest dietary level. In the biochemical studies, reductions in the blood urea levels occurred in both sexes and were significant in all groups except at the 0-06 and 0'6~0 levels. The uneven group distribution of this effect suggests that a cause other than Chocolate Brown HT is likely. Increases in total serum protein were seen in males on the 0"6 and I ~o levels. The absolute weights of the heart, kidneys, liver, spleen and testes remained unaffected at all levels. There was no evidence of pathological damage at any dietary level of the colouring administered but pigment was seen at the two highest levels in certain intestinal cells, lymph nodes and cells of the proximal convoluted tubes of the kidney. On the basis of these results a maximum no-effect level of Chocolate Brown .HT has been established at 0"6 ~ in the diet of rats for 90 days. INTRODUCTION I n c o n j u n c t i o n w i t h t h e s h o r t - t e r m study o f C h o c o l a t e B r o w n H T in the r a t r e p o r t e d in the previous p a p e r (Hall, Lee & F a i r w e a t h e r , 1966) a similar study was u n d e r t a k e n with a view to c o m p a r i n g t h e results o b t a i n e d b y two i n d e p e n d e n t g r o u p s o f w o r k e r s e m p l o y i n g p r o c e d u r e s t h a t were essentially similar b u t differed in p o i n t s o f detail. T h e status o f C h o c o late B r o w n H T as a f o o d c o l o u r i n g in t h e U K has been outlined b y H a l l et al. (1966), w h o also n o t e d t h a t t h e r e were no p u b l i s h e d d a t a on the toxicity o f this colouring. EXPERIMENTAL
Materials. T h e s a m p l e o f C h o c o l a t e B r o w n H T used in t h e s h o r t - t e r m study in rats was supplied b y t h e same source a n d h a d t h e same chemical specification as described by H a l l et al. (1966). Animals. W e a n l i n g C a r w o r t h F a r m rats r e a r e d in t h e T u n s t a i l L a b o r a t o r y b r e e d i n g unit were used. Experimental design and conduct. G r o u p s o f 12 rats, equally divided b y sex a n d h o u s e d i n d i v i d u a l l y in cages, were m a i n t a i n e d on diets c o n t a i n i n g C h o c o l a t e B r o w n H T at levels o f 0.02, 0.06, 0-20, 0.60, 1.0 a n d 2.0 ~ for 90 days. A c o n t r o l g r o u p consisting o f 24 m a l e a n d 24 female rats received t h e basic diet for t h e s a m e p e r i o d . F o o d ( p o w d e r e d 86 diet) a n d Water were supplied ad lib. B o d y weight was r e c o r d e d weekly a n d f o o d c o n s u m p t i o n twice weekly. H a e m a t o l o g i c a l studies c o n d u c t e d t e r m i n a l l y involved m e a s u r e m e n t s o f h a e m o g l o b i n , t o t a l red a n d white cell counts a n d h a e m a t o c r i t . S e r u m levels o f p r o t e i n a n d u r e a were
151
152
P.L. CHAMBERS, C. G. HUNTER a n d D. E. STEVENSON
also determined. A t autopsy, the heart, liver, spleen, kidneys a n d testes were weighed a n d submitted to m a c r o p a t h o l o g i c a l e x a m i n a t i o n , t o g e t h e r with the o t h e r m a j o r organs. Paraffin sections o f the m a j o r organs, stained with h a e m a t o x y l i n a n d eosin, were exa m i n e d histologically. RESULTS A l t h o u g h some rats h a d m i n o r r e s p i r a t o r y d i s o r d e r s the r e m a i n d e r in all the g r o u p s r e m a i n e d healthy. Ten deaths were recorded, but these were evenly d i s t r i b u t e d t h r o u g h o u t the c o n t r o l and test groups. T h e rats grew well a n d the m e a n final b o d y weights showed no significant change (Table 1). However, t h e non-significant reductions in weight gain at t h e 1 a n d 2 % levels b e c a m e significant when account was t a k e n o f the fact t h a t the g r o u p s involved c o n s u m e d m o r e f o o d t h a n their respective controls a n d an a p p r o p r i a t e c o r r e c t i o n i n t r o d u c e d to m a k e allowance for the lowered efficiency o f f o o d utilization at the higher d i e t a r y levels (Table 1). H a e m a t o l o g i c a l e x a m i n a t i o n revealed slight, b u t nevertheless significant, reductions in h a e m o g l o b i n , red cell c o u n t a n d p a c k e d cell v o l u m e in males on the 2 % level (Table 2). In the biochemical studies, t o t a l serum p r o t e i n was increased in m a l e rats on the 0.6 a n d 1.0 % levels b u t n o t at 2 ~o o r any o t h e r level. F e m a l e g r o u p s r e m a i n e d unaffected (Table 3). The b l o o d u r e a c o n c e n t r a t i o n was depressed in each test g r o u p o f b o t h sexes a n d with the exception o f rats on 0.06 a n d 0.6 % o f the c o l o u r i n g the reductions were significant (Table 3). There were no i m p o r t a n t changes at any level in the a b s o l u t e weights o f the heart, kidneys, liver, spleen and testes (Table I). Table I. E~rectof 90-day feeding of 0-2 % Chocolate Brown HT on growth, food consumption and organ weights of rats Mean group Dietary terminal body weight level (~0) Uncorrected Corrected'l-
Total food intake (g)
Heart
Liver
Spleen
Kidneys
Testes
13.26 14.13 12-27 12-63 13.38 12.29 11-72
0-84 0.79 0.75 i.005 0.78 0.73 0-95
2-17 2-28 2-14 2-08 2.35 2.24 2.14
3"32 3"18 3"23 3'41 3"22 3"26 3"50
8"32 7-98 8-42 7"85 8'02 8"26 7-82
0"62 0'62 0"61 0-60 0.60 0"69 0"65
1"44 1"38 1-45 1"34 1"41 1"47 1-39
Absolute organ weight (g)
0.0 0.02 0.06 0.2 0.6 1.0 2.0
343 357 332 333 337 326 321
344 350 344 341 337 324* 303***
1962 2017 1903 1924 1970 1984 2082
Males 1-09 1-11 1.05 1.04 1-09 1.09 1.07
0"0 0"02 0'06 0"2 0"6 1"0 2-0
224 217 226 217 222 226 211
223 225 226 223 223 218 204**
1636 1505 1630 1527 1612 1752 1736
0"81 0-81 0"82 0'77 0"76 0'85 0"76
Females
Values marked with asterisks differ significantly from those of controls: *P<0"05; **P
S H O R T - T E R M TOXICITY OF C H O C O L A T E B R O W N H T
8
&
8
~~-~-
V
-
'~ I ~
e,i F',
±~,-=
8
o
'~
E "~ --~..2, x x,.= ~. o o -a(O~
>,~
o
153
154
P.L. CHAMBERS,C. G. HUNTER and D. E. STEVENSON Table 3. Blood urea and serum protein levels in rats fed Chocolate Brown H T at dietary levels o! O--2% for 90 days
Dietary level (%)
Blood urea (mg/100ml)
0'0 0.02 0"06 0'2 0"6 1"0 2'0
Males 42.65 37.18"* 39-25 36"40*** 39"36 35"18"#* 35"30***
0'0 0'02 0"06 0"2 0"6 1"0 2'0
42'30 38"91" 40"18 35"54*** 41"00 35-00*** 36'80
Total serum p r o t e i n (g/100ml)
•
6'50 6"45 6"63 6"66 6"72* 6"74* 6"49
Females
6-65 6"53 6"67 6'65 6.64 6"40 6.73
Values marked with asterisks differ significantly from those of controls: *P
SHORT-TERM TOXICITY OF CHOCOLATE BROWN I-IT
155
REFERENCES Hall, D. E., Lee, F. S. & Fairweather, F. A. (1966). Acute (mouse and rat) and short-term (rat) toxicity studies on Chocolate Brown HT. Fd Cosmet. Toxicol. 4, 143. E t u d e s ~ court terme de l'action du colorant Chocolat Brun H T c h e z le rat R~sum~---Des rats ont 6t(~ soumis ~. des r~gimes alimentaires contenant 0,0; 0,02; 0,06; 0,20; 0,60; 1 et 2 % de colorant Chocolat Brun HT durant une p6riode de 90 jours. Aucune action d~favorable n'a 6t6 o b s e r v ~ sur l'6tat g~n6ral, sur le comportement ou sur la survie de ces animaux. Bien que le gain pond6ral chez les rats soumis b. ces r6gimes n'ait pas diff6r6 d'une fad;on nette de celui des t6moins, cependant, s i r o n tient compte des ingesta totaux, le 16get retard de croissance constat6 chez les animaux des deux sexes recevant des rb~gimescontenant 1 et 2 % de colorant, s'est av6r6 significatif. Chez les rat miles soumis ~. des r(~gimes contenant le pourcentage le plus 61ev6 de colorant, des examens h6matologiques ont montr6 une baisse l~g~re mais nette du taux d'h~moglobine, du nombre des globules rouges et de l'h6matocrite. Les ~preuves biologiques ont montr6 une baisse de l'ur~e sanguine chez les animaux des deux sexes, marqu6e chez tousles groupes saul ceux soumis b. des r6gimes contenant 0,06 et 0,6 % de colorant; le caract~re irr6gulier de cette baisse parmi les divers groupes laisse penser qu'un facteur autre que le colorant Chocolat Brun HT a 6t6 en cause. Chez les rats mfiles soumis ~. des r~gimes contenant 0,6 et 1% de colorant, il a 6t6 constat6 une augmentation de la prot6inimie. Le poids absolu du coeur, des reins, du role, de la rate et des testicules n'a pas 6t6 affect6 darts tousles groupes. Aucun signe de 16sion pathologique n'a ~t~ constat6 chez les rats des diff6rents groupes recevant le colorant, si ce n'est la pr6sence de pigments au niveau de certaines cellules intestinales, des glandes lymphatiques et des cellules des tubes contourn~s du rein chez les animaux soumis aux r~gimes contenant les pourcentages les plus 61ev6s de colorant. Selon les r~sultats de ces 6tudes, il a 6t(~ 6tabli que 0,05 % de colorant Chocolat Brun HT administr(~ au rat durant 90 jours n'entraine aucune action d~favorable. K u r z z e i t i g e Untersuchung der Wirkung yon Schokoladenbraun H T a u f R a t t e n Zusammenfassung--Schokoladenbraun HT wurde 90 Tage lang an Ratten in Konzentrationen von 0,0; 0,02; 0,06; 0,20; 0,60; 1,0 und 2% im Futter verabreicht. Es wurde keine nachteilige Wirkung hinsichtlich des Aussehens, des Verhaltens und des ~berlebens der Tiere beobachtet. Wenn auch bei Anpassung der Gesamtfuttermenge die Zunahme des K6rpergewichts der behandelten Ratten sich nicht signifikant yon derjenigen der Kontrolltiere unterschied, so wurde doch eine leichte Wachstumsverz6gerung bei den m~nnlichen Tieren, die 1 und 2% erhielten, und bei weiblichen Tieren der h6chsten Konzentrationsgruppe signifikant. Die h~.matologische Untersuchung zeigte leichte, aber signifikante Verminderungen des H~.moglobins, der Erythrozytenzahl und des H§.matokrits bei m~.nnlichen Ratten mit der h6chsten Konzentration im Futter. Bei den biochemischen Untersuchungen wurden Verminderungen des Harnstoffgehalts des Blutes bei beiden Geschlechtern beobachtet, und sie waren in allen Gruppen ausser denen mit einer Konzentration yon 0,06 und 0,6 % im Futter signifikant. Die ungleichm~ssige Gruppenverteilung dieses Effekts l~.sst vermuten, dass eine andere Ursache als das Schokoladenbraun HI" dafi~r verantwortlich ist. Eine Zunahme des Gesamtserumproteins wurde bei m~.nnlichen Tieren beobachtet, die 0,6 und 1% erhielten. Die absoluten Gewichte yon Herz, Nieren, Leber, Milz und Hoden blieben bei allen Konzentrationen unbeeinflusst. Es gab keine Anzeichen pathologischer Sch~.den bei irgendeiner Konzentration des Farbstoffs, jedoch wurde bei den beiden h6chsten Konzentrationen Pigment in bestimmten intestinalen Zellen, Lymph Knoden und Zellen der proximalen gewundenen Harnkan~ilchen der Niere festgestellt. Auf der Grundlage dieser Ergebnisse wurde eine maximale wirkungslose Dosis von Schokoladenbraun H T mit 0,5 ~o im Futter von Ratten ftir die Dauer von 90 Tagen festgesteUt.