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Toxicology, 8 (1977) 387--390 © Elsevier/North-Holland Scientific Publishers Ltd.
Let ter to the Editors
TOXICOLOGICAL ASSESSMENT OF FLAVOURING ESTERS
We have noted with interest the publication in Toxicology of the paper by Longland et al. [1] dealing with the use of in vitro hydrolysis procedures as a basis for determining the necessity for further toxicological studies of flavouring esters. It is worth reiterating that the Joint FAO/WHO Expert Commit tee on Food Additives [2] have given the opinion that if evidence can be produced to show that an ester is readily hydrolysed by mammalian enzymes to consti tuents whose metabolic fate and biological interactions are fully understood, further toxicological studies may not be necessary.
We are aware of both national and international activities relating to the regulation of flavouring substances and of the need to provide biological information in this context . In order to avoid possible duplication of experi- mental effort we take this oppor tuni ty to bring to your attention the find- ings of preliminary investigations undertaken under the auspices of the International Organization of the Flavour Industry (IOFI). The studies, which were carried out independently at the laboratories of CIVO/TNO, Holland [3 ] , were designed to establish the relative rates of enzymic hydro- lysis of 26 esters of organic alcohols and acids of various structural configu- rations, chosen on the bases summarized in Table I.
Initial investigations used a preparation of pancreatin (Merck 7132). Incubations were made under arbitrary experimental conditions of pH, enzyme concentrat ion and substrate concentrat ion over 2 h at 37 ° C. The extent of hydrolysis was determined by gas--liquid chromatography.
Table II records the percentage hydrolysis of each ester under the con- ditions of the experiment. It will be noted that in the majority of cases, the selected esters were hydrolysed rapidly by pancreatin, although in several instances hydrolysis was low or undetectable.
For this reason additional investigations were undertaken on 8 esters in the latter category using whole homogenates of pig je junum and liver. Incubations were again made under arbitrary conditions of pH, enzyme concentrat ion and substrate concentration and hydrolysis after 2 h was measured as before using gas--liquid chromatography. The results are sum- marized in Table III.
It will be seen from the foregoing that, in agreement with the findings of Longland et al. [1] , there was wide variation in the degree of in vitro hydro- lysis o f flavouring esters depending on the source of mammalian enzymes
387
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TABLE II
HYDROLYSIS OF FLAVOURING ESTERS BY PANCREATIN
Incubations were made in 0.5 M phosphate buffer at pH 7.5 and 37 ° C. The extent of hydrolysis was determined after 2 h by gas--liquid chromatography of the ester.
Ester Conc. a GLC column used for % Hydro- (~1/1) lysis
Ester Alcohol
iso-Amyl furylpropanoate 27 Carb 20 M (2) Ethyl cyclohexylpropanoate 36 Carb 20 M (2) AUyl hexanoate 60 Carb 400 Benzyl 2-methylbutanoate 40 Carb 20 M (1) m-Cresyl phenylacetate < 9 mg/1 ef Dexsil 300 Citronellyl acetate 15 Carb 400 Benzyl phenylacetate 36 Dexsil 300 Ethyl furylpropanoate 40 FFAP Allyl phenylacetate 50 FFAP iso Amyl phenylacetate < 25 f Carb 20 M (2) Ethyl decanoate < 15 f Carb 400 Benzyl cinnamate 18 mg]l e Dexsil 300 Methyl phenylacetate 100 Carb 20 M ( 1 ) o-Cresyl acetate 70 Carb 20 M (1) Citronellyl phenylacetate < 18 Dexsil 300 Benzyl acetate 70 Carb 20 M (1) iso Propyl phenylacetate 90 Carb 20 M (2) iso Propyl butanoate 180 Carb 400 iso Amyl acetate 100 Carb 400 1,3-Dimethylbutyl acetate 100 Carb 400 Ethyl acetate 90 Carb 400 Benzyl tiglate < 25 ~ Carb 20 M (1) Allyl tiglate 100 Carb 400 Methyl anthranilate 100 Carb 20 M (1) Methyl-N-methylanthranilate 15 FFAP Dimethylbenzylearbinyl acetate 65 Carb 20 M (2)
Carb 400 100 Carb 400 100 Carb 400 100 Carb 20 M (1) 100 Dexsil 300 100 Carb 20 M (2) 100 Carb 20 M (2) 100 Carb 400 100 Carb 400 100 Carb 400 90b; Carb 400 80 Carb 20 M (2) 80 Carb 400 70 Carb 20 M (1) 60 Carb 20 M (2) 60 Carb 20 M (1) 50 Carb 400 50 Carb 400 40 Carb 400 20 Carb 400 15 Carb 400 _d Carb 20 M (1) _d Carb 400 __d Carb 400 _d Carb 400 --d Carb 20 M (2) --d
100 c
a Concentration in incubation mixture in ulfl unless otherwise stated. b 2-Methylbutyl phenylacetate. c Amyl phenylacetate. d Not measurable. e Esters dissolved in ethanol initially and then diluted with water. f Estimated concentrations.
u sed . M o r e o v e r , m a n y o f t h e es ters w h i c h a p p e a r t o res is t t h e a c t i o n o f p a n c r e a t i n w e r e h y d r o l y s e d r e a d i l y b y f resh h o m o g e n a t e s o f m a m m a l i a n t issues . I n t h e a b s e n c e o f e v i d e n c e t o t h e c o n t r a r y i t m u s t b e p r e s u m e d t h a t t h o s e es ters h y d r o l y s e d r a p i d l y in v i t r o wil l a lso be d e g r a d e d r e a d i l y in t h e i n t a c t a n i m a l .
T h e r e is a p a u c i t y o f p u b l i s h e d i n f o r m a t i o n o n t h e in v ivo m e t a b o l i s m o f f l a v o u r i n g es ters b y m a m m a l s . C o n s e q u e n t l y , I O F I has c o m m i s s i o n e d f u r t h e r
3 8 9
TABLE III
HYDROLYSIS OF FLAVOURING ESTERS BY WHOLE HOMOGENATES OF PIG JEJUNUM AND LIVER
Incubations were made in 0.5 M phosphate buffer at pH 7.5 and 37 ° C. The extent of hydrolysis was determined after 2 h by gas--liquid chromatography of the ester.
Ester Conc. GLC column used for % Hydrolysis (~1/1)
Ester Alcohol Jejunum Liver
iso-Amyl acetate 500 Carb 400 Carb 400 100 _ a Ethyl acetate 400 Carb 400 Carb 400 100 _ a Allyl tiglate 400 Carb 400 Carb 400 100 __a 1,3-Dimethylbutylacetate 400 Carb 400 Carb 400 > 99 __a Benzyl tiglate 400 SE 30 SE 30 90 _ a Methyl-N-methylanthranilate 250 SE 30 Cart) 400 20 > 99 Methyl anthranilate 250 SE 30 Carb 400 15 > 99 Dimethylbenzylcarbinyl
acetate 250 SE 30 SE 30 0 20
a Extent of hydrolysis was not measured in these instances.
investigations on 4 esters selected on the basis of the studies above and representing classes which are hydrolysed readily (isoamylfuryl propionate and allyl phenylacetate), relatively slowly (methyl-N-methylanthranilate) or negligibly (dimethylbenzylcarbinyl acetate) under in vitro conditions. These studies using a procedure permitting the measurement of both the intestinal absorption of esters and comparison of their relative rates of hydrolysis in the guinea pig [4] will form the subject of a future report [5].
Dr. F. Grundschober Scientific Adviser o f IOFI
REFERENCES
1 R.C. Longland, W.H. Shilling and S.D. Gangolli, Toxicology, 8 (1977) 197. 2 Joint FAO/WHO Expert Committee on Food Additives, l l t h Report, Specifications
for the identity and purity of food additives and their toxicological evaluation. Some flavouring substances and non-nutritive sweetening agents. W.H.O. Tech. Rep. Ser. No.383, 1968.
3 D.C. Leegwater and S. van Straten, Unpublished CIVO/TNO Reports Nos. R4319 and R4414.
4 K.R. Butterworth and D. Pelling, J. Physiol., 232 (1973) 60. 5 D. Pelling, R.C. Longland, M. Dulley and S.D. Gangolli, Toxicology, submitted.
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