FdCosmet. Toxicol. Vol. 3, pp. 145-147. Pergamon Press 1965. Printed in Great Britain
SUMMARIES OF TOXICOLOGICAL DATA
ARTIFICIAL SMOKE FLAVOURINGS
Summaries* of two unpublished reports on the composition (I4/. LUinsky & P. Shubik, 1962) and toxicological assessment (Woodard Research Corporation, 1963) of liquid smoke.
Introduction The traditional method of preparing smoked foods involves the exposure of the food to
wood smoke for a certain length of time. Recent investigations have revealed, however, that the process is not without hazard so far as the consumer is concerned in view of the presence of the carcinogenic hydrocarbon 3,4-benzopyrene (BP) in smoked meat and fish products (Cited in F.C.T. 1963, 1, 118) and in smoked haddock and salmon (see p. 130 of this issue). Indeed, some considerable effort has been exerted in attempts to eliminate BP by modifying the conditions of the process (Cited in F.C.T. 1963, 1,279; ibid 1964, 2, 255) development of liquid smokes, produced by water absorption of wood smoke from which tarry droplets have been removed by filtration, constitutes a rather more precise method of smoking than the traditional one. The present findings that the liquid smoke called SF-12 is devoid of BP, and exercises no toxic effect when fed to laboratory animals for 90 days represent a unique collaborative achievement from the technological and toxicological viewpoints.
The first of two unpublished reports summarized below deals with the identification and estimation of polycyclic aromatic hydrocarbons in Liquid Smoke SF-12. In the second report the results are presented of 90-day toxicity studies carried out on Liquid Smoke SF-12 and "CharSol".
I. The Detection and Estimation of Polycyclie Aromatic Hydrocarbons in Liquid Smoke
W. LIJINSKY and P. SHUBIK'[" Division of Oncology, Chicago Medical School, Institute of Medical Research, Chicago 12, IlL, USA
(Report dated 12 December 1962)
Source of sample The sample of Liquid Smoke SF-12 was obtained from the Griffith Laboratories Inc.,
Chicago, II1., USA.
Analytical procedure Benzene extracts of 4 1. Liquid Smoke SF-12 were reduced in vacuo to a residue (78 g)
which was taken up in methanol/cyclohexane. The concentrate of the cyclohexane fraction was then partitioned between cyclohexane and nitromethane and the nitromethane fraction distilled in vacuo to yield a residue (5-9 g) which was chromatographed on a silica gel
*Prepared by B1BRA and published with the permission of T. Lucas & Co. Ltd., Kingswood, Bristol, England.
t Toxic. appl. Pharmac. In press.
146 SUMMARIES OF TOXICOLOGICAL DATA
column developed initially with hexane--cyclohexane, then with hexaae-benzene and finally with benzene. The pooled residues obtained on evaporating the two mixed eluates constituted Fraction A (>100 mg) while the residue of the evaporated benzene eluate represented Fraction B (300 mg).
Identification and estimation of polycyclic aromatic hydrocarbons Fractions A and B were subjected to descending paper chromatography using 2,2,4-
trimethylpentane as the developing solvent. Fraction B resolved into 7 fluorescent bands whose ultraviolet spectra did not indicate the presence of polycyclic aromatic hydrocarbons. A tentative identification of only one compound, carbazole, was made and no further work was conducted on this fraction.
Fraction A gave 5 distinct fluorescent bands which were rechromatographed on acety- lated paper using, toluene-methanol-water (1:10:1, by vol.) as the developing solvent.
These bands resolved into further bands which were examined by ultraviolet absorption and fluorescence emission spectroscopy. The spectra of one band indicated the presence of benz[a] anthracene and chrysene; another band, the presence of pyrene and fluoranthene. Although ultraviolet spectra suggested the presence of BP and benzo[g,h,i,]perylene both possibilities were ruled out by their fluorescence emission spectra.
As determined by ultraviolet spectroscopy, the concentrations in ppm of the non- carcinogenic hydrocarbons tentatively identified in Liquid Smoke SF-12 were: benz[a]- anthracene (0.012); carbazole (0.200); chrysene (0.011); pyrene (0.013); fluoranthene (0.032).
Confirmatory evidence for absence of BP in SF- 12 The presence of BP in the Liquid Smoke SF-12 would not have gone undetected, as
evidenced by a positive control experiment in which BP was added to a sample of SF-12 at 0.012 ppm. Repetition of the extraction and part of the chromatographic procedures showed that 73 % of BP was recovered from Fraction A and furthermore the fluorescent band containing BP--which did not appear in the test sample--was clearly distinguishable from all the other components in this fraction.
II. "Liquid Smoke" Flavor: Safety Evaluation by Oral Administration to Rats for 90 Days
(Report submitted by tile Woodard Research Corporation, Herndon, Va., USA, to the Griffith Laboratories Inc., Chicago, IlL, USA, and dated 2 April 1963)
Subacute toxicity studies 90-Day toxicity studies in rats were carried out on samples of Liquid Smoke SF-12 and
"CharSol"* obtained from the Griftith Laboratories Inc. Groups of 25 male and 25 female rats received 0, 0.25 and 2.0 % Liquid Smoke SF-12 in
~*According to Hollenbeck & Marinelli (Proc. 15th Res. Conf. Am. Meat Inst. Found. Univ. Chicago, 1963, Circular No. 74, p. 67) "CharSol" is the name given to the smoke flavour solution obtained by con- centrating the soluble constituents of water into which the vapour phase of hard wood smoke has passed. By means of fractionation and paper chromatographic techniques the following major compounds were found present in "'CharSol": formic, propionic, vanillic and syringic acids; dimethoxyphenol; methyl glyoxal; furfural; acetaldehyde; acetone; ethanol and BP. Reference has already been made (Cited in F.C.T. 1963, 1, 280) to the identification in curing smoke of acetic and formic acids (70% of organic aci:l content of smoke); vanillin; ethylvanillin ; vanillic acid; syringic aldehyde and its corresponding acid and fl-resorcylic acid.
SUMMARIES OF TOXICOLOGICAL DATA 147
their diet. These groups will be referred to as groups I, II and HI respectively. A positive control group (group IV) received "CharSol" at a level of 2 ~o of the diet. Food and water were supplied ad lib.
Growth and food consumption. Weekly measurements of weight gain and food intake of animals of all test groups did not reveal any difference from the control group.
Behaviour and general condition. Apart from rats of group IV which occasionally exhibited signs of hypersensitivity to external stimuli the behaviour and general condition of all other animals in both test and control groups were normal.
Haematology. Haemoglobin, haematocrit and total and differential white cell counts, which were determined on 5 males and 5 females each from groups I, IH and IV at weeks 6 and 13, did not depart from normal limits.
Mortality. With the exception of one female of group II which died during week 3 all animals survived the 12-week period.
Organ weights. Terminal absolute and relative weights of the heart, liver, kidney, spleen, testis or ovary and adrenals were comparable in all groups.
Histopathology. Some 20 major organs and tissues were examined in each of 37 rats-- 5 males and 5 females of groups I, III and IV; and in 7 other animals (4, 2 and 1 from groups II, III and IV respectively) because of death or gross abnormalities (unspecified) seen at necropsy. The incidence of distinct minor degenerative changes of the liver and kidney was somewhat higher in rats of group III as compared with other groups. In rats of groups III and IV there was a tendency towards slight bone marrow hyperplasia although in neither instance were these effects considered to be outside normal limits. No other signifi- cant micropathological finding was reported.
Summary and Conclusions
3,4-Benzopyrene which has been found present in certain smoked products was not detectable in Liquid Smoke SF-12. Five non-carcinogenic polycyclic aromatic hydrocarbons, benz[a]anthracene, carbazole, cllrysene, pyrene and fluoranthene were tentatively identified; on the basis of ultraviolet spectral measurements they were present at levels of 0.012, 0.200, 0.011, 0.013 and 0.032 ppm in Liquid Smoke SF-12.
From the subacute toxicity studies the conclusion was drawn that: "Both Liquid Smoke and CharSol at the levels fed were essentially devoid of any signs of toxicity. No histo- pathological changes were seen in the experimental rats that were not also seen in the con- trois. The frequency of these changes was somewhat greater, however, in the experimental groups."
[Editor's Note: The two Reports summarized above and other data were submitted by The Griffiths Laboratories Inc. to the US Department of Agriculture (USDA). After reviewing this and other available information the USDA declared that Liquid Smoke SF-12 was acceptable for use in accordance with the regulation permitting the use of smoke flavourings in meat food products.]