Fd Chem. Toxic. Vol. 24, No. 10/I 1, pp. 1063-1065, 1986 0278-6915/86 $3.00 + 0.

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Printed in Great Britain Pergamon Journals Ltd

TOXICOLOGY OF tert-BUTYLHYDROQUINONE (TBHQ)

Abstract--This paper traces the history of the EEC Scientific Committee for Food's examination of
available toxicological data on tert-butylhydroquinone (TBHQ). Studies of mutagenicity, metabolism,
enzyme activities, reproduction and long-term toxicity and carcinogenicity are reviewed. The author
concludes that dose level is an important factor in the induction of forestomach tumours. Further
long-term toxicity testing of TBHQ is needed to evaluate its genotoxicity. However, since information is
not available, an attempt is made to calculate the 'safety' margin between actual intake by man and the
conventional no-effect level in dogs and rats.

The Joint FAO/WHO Expert Committee on Food experiments are necessary to clarify this point before
Additives (JECFA) evaluated tert-butylhydro- TBHQ can be considered acceptable for use in food
quinone (TBHQ) in 1975 and 1978 and established a in the EEC.
group acceptable daily intake (ADI) of 0-0.5 mg/kg Relevant to the evaluation are metabolic data
body weight for man for BHA, BHT and TBHQ. derived from both animals and man. Metabolic stud-
TBHQ alone or in combination with BHA and/or ies in the rat and dog showed rapid absorption from
BHT is cleared in the US provided that the total the gastro-intestinal tract and rapid excretion (up to
antioxidant content of the food does not exceed 80% in the first 24 hr) in the urine, primarily as the
0.02% of the oil or fat content (21 CFR 172.185). The 4-O-sulphate conjugate, with smaller quantities of
levels that are cleared by the US Meat and Poultry the 4-O-glucuronide conjugate and minor quantities
Inspection Division range from 0.003% to 0.02% for of unchanged TBHQ. In a long-term study with rats,
the various meat products. In margarine a level of approximately 65% 4-O-sulphate and approximately
0.02% is considered acceptable in fat and oil, alone 30% 4-O-glucuronide were found in the urine
or in combination with BHA and/or BHT (Federal (Astill, Blakely, Cantor et al. 1968; Astill, Cantor &
Register, 22 November 1983). In addition to the McEwan, 1967). When human subjects received
USA, nine countries have accepted the use of TBHQ TBHQ in oil or in gelatin capsules, examination of
as an antioxidant. the urine indicated that TBHQ was excreted as the
The status of TBHQ in Europe, in particular in the O-sulphate and O-glucuronide conjugates (in a ratio
European Economic Community (EEC), is different. of approximately 3:1). No unchanged TBHQ was
At the time a positive list of antioxidants was com- detected (Astill, Cantor, Ely et al. 1967).
posed, the countries of the EEC were not interested TBHQ, like BHA and BHT, has an influence on
in placing TBHQ on the list, probably because this enzyme activity in rat-liver microsomal fractions.
antioxidant was not used. In 1981, the EEC Scientific However, the effect of TBHQ is weaker than that of
Committee for Food (SCF) was asked to evaluate the BHA and BHT (Tischer & Walton, 1968). In a
available toxicological information on TBHQ in or- 180-day study with rats and a 2-yr dog study
der to advise the EEC Commission. At that time, the (117wk), a dose level of 0.5% TBHQ in the diet did
SCF concluded that it was not possible to establish not cause significant changes in enzyme activities in
an ADI until further information on genotoxicity the liver (Food and Drug Research Laboratories
became available. (FDRL), 1968).
In 1984 and 1985, TBHQ was re-evaluated when The 117-wk dog study showed only minor changes.
data became available on in vivo and in vitro Red blood cell counts were slightly lower in dogs of
mutagenicity and on the similarity between the action the highest dose group (0.5%) than in controls. These
of TBHQ and BHA on the rat forestomach. The SCF changes were also reflected in the haemoglobin con-
concluded that the genotoxic potential of TBHQ tent and haematocrit values. However, these effects
could not be excluded. Therefore, further animal were not significant and were not observed at the
lower dose levels. There was a tendency toward
increased liver weight at the highest dose level, but
*Formerly of the National Institute of Public Health and EM examination of liver and kidneys did not show
Environmental Hygiene, Bilthoven, The Netherlands.
any abnormalities. It appears that in the dog the
Abbreviations: ADI =acceptable daily intake; BHA=
butylated hydroxyanisole; BHT = butylated hydroxy- no-effect level is 0.5% TBHQ in the diet (FDRL,
toluene; FDRL = Food and Drug Research Laborato- 1968; Wolf & Fassett 1968a, b).
ries, Inc.; JECFA = Joint FAO/WHO Expert Commit- Three rat reproduction studies, one over three
tee on Food Additives; SCF = ScientificCommittee for generations, have been carried out. The highest dose
Food; TBHQ = tert-butylhydroquinone. level tested was 0.5% TBHQ in the diet. At this dose
1063
 1064 G.J. VAN ESCH

level food intake was decreased, with a consequent seem to be without effect. Iverson, Lok, Nera &
decrease in the body weight of the pups. Palatability Clayson (1985) found that the high rate of cell
may have played a role in the decreased food intake. proliferation in the rat forestomach appears to be
In the teratogenicity studies with rats, no visceral or dependent on the continued presence of BHA.
skeletal abnormalities were found with dose levels up Removal of BHA from the diet leads to a complete
to 0.5% TBHQ (Fassett, Roudabush & Terhaar, and rapid regression of the excessive level of cell
1965; Krasavage, 1977; Krasavage & Terhaar, 1970; proliferation and a much slower regression of the
Terhaar & Krasavage, 1968). pathological lesions. They suggest that "BHA is
The available mutagenicity studies show that acting almost entirely as an inducer of cell prolif-
the Ames test (using five strains of Salmonella eration rather than as an initiating agent capable of
typhimurium with and without metabolic activation) altering cells".
was negative, the V79 Chinese hamster cell test was Available studies indicate that the dose level is an
positive, the micronucleus test in mice showed one important factor in the induction of forestomach
negative and one positive, the cytogenetic study using tumours. Whether, in this process of proliferation, a
bone-marrow cells of rats was positive, the L5178Y no-effect level exists, remains unknown.
mouse lymphoma cell test was positive, the DNA For TBHQ, the available data are inadequate to
metabolic test using spleen cells from Chinese ham- exclude genotoxicity. Because the compound is used
sters was equivocal and the dominant lethal test in in a number of countries, it would be valuable to
rats was negative (CERTI, 1982; Giri, San, Talukdor calculate the 'safety' margin between actual intake by
et al. 1984; IPL 1982a, b; Krasavage, 1984; Litton man and the no-effect level in dogs and rats.
Bionetics, Inc., 1982a, b; Mueller & Lockhart, 1983; The daily intake of TBHQ in the USA is expressed
OEFZS, 1983). Additional information shows that as the 'per capita daily intake' (PCDI) (an over-
the CHO/HGPRT forward mutation assay and the estimation), which is 14 mg/person/day and as the
mouse bone-marrow (male ICR mice) cytogenetic 'potential daily intake' (PDI), which is
assay were negative (Health and Environment Labor- 0.42 mg/person/day (Flamm, Weisburger, Clayson et
atories, 1985; Litton Bionetics, Inc., 1985). Of these al. (1982). When we compare these intake figures with
nine tests, only three were positive. the no-effect level (on which the JECFA ADI is
A long-term toxicity study was conducted in an based), it is clear that the margin of safety (using the
attempt to gain a better perspective regarding the actual intake of 0.42 mg) is at least 7000. On the basis
short-term genotoxicity tests (Terhaar, Krasavage, of the rat studies, this margin of safety would be
Wolf & Leonard, 1968). This study was carried out approximately 15,000. However, the genotoxicity of
with albino rats from A and C Farms, using TBHQ TBHQ cannot be resolved on the basis of the avail-
dose levels of 0, 0.016, 0.05, 0.16 and 0.5% for 20 able data, and this potential effect needs to be
months. Study parameters included food intake, explored further. In the meantime, the FDA has
growth, feed efficiency, haematological and biochem- placed TBHQ on the list of priority chemicals for
ical tests, urinalysis, organ weights and extensive testing by the National Toxicology Program (NTP).
histopathology. None of the treatment groups Until these studies become available, I am of the
differed significantly from the controls. During the opinion that TBHQ used at the present levels is safe.
12-20-month investigation, however, mortality was Re-evaluation should be carried out within the next
high and, as a consequence, the number of animals 3-5 years.
remaining at the end of 20 months was small. The
SCF concluded that this study did not address the
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