TBHQ (TERT-BUTYLHYDROQUINONE)
EXPLANATION
TBHQ was evaluated for acceptable daily intake for man by the
Joint FAO/WHO Expert Committee on Food Additives at its nineteenth and
twenty-first meetings (Annex 1, references 38 and 44). A toxicological
monograph was issued after the nineteenth meeting (Annex 1,
reference 39). A group ADI for TBHQ, BHA, and BHT of 0-0.5 mg/kg b.w.
was established at the twenty-first meeting. Since the previous
evaluation, additional data have become available and are summarized
and discussed in the following monograph addendum.
BIOLOGICAL DATA
Toxicological studies
Special study on effect on prothrombin time
Rats
Male albino rats (CRL:COBS.CD(SD)BR) were dosed daily (i.p.) on 3
consecutive days with TBHQ at dose levels equivalent to 50, 100, or
150 mg/kg b.w. No spontaneous haemorrhage was observed in any of the
test animals on autopsy, nor was there any effect on prothrombin time.
It should be noted that in a similar study in this strain of rats, BHT
at dose levels up to 1520 mg/kg b.w. failed to produce haemologic
deaths, and only at this high dose did BHT cause a significant
increase in prothrombin time (Krasavage, 1984).
Special studies on mutagenicity
Ames Salmonella/microsome bacterial mutagenesis test
TBHQ was non-mutagenic in the Salmonella typhimurium plate
incorporation assay using bacterial strains TA98, TA100, TA1535,
TA1537, and TA1538, both in the absence and in the presence of a
metabolic activation system derived from rat liver S-9 fraction. Doses
tested were up to 5.0 µg/ml for strain TA1535, 15 µg/ml for strain
TA1537, and 50 µg/ml for strains TA98, TA100, and TA1538 (Société
Kemin Europa, 1982a).
In another study using these bacterial strains, with and without
a metabolic activation system derived from Arochlor 1254-induced
liver, no indication of mutagenicity was seen at test levels up to
0.45 mg/plate in the absence of S-9 fraction and up to 2.7 mg/plate in
the presence of S-9 fraction (Mueller & Lockhart, 1983).
Mouse lymphoma forward mutation assay
TBHQ was active in the mouse lymphoma forward mutation assay
using mouse lymphoma cell line L5178Y (TK+/-) with and without a
metabolic activation system (S-9 fraction prepared from
Arochlor-induced Sprague-Dawley rats). The highest concentration of
TBHQ tested (31.3 ng/l) induced a mutant frequency that just exceeded
the minimum criterion for considering this a positive test. With
metabolic activation, a dose-dependent increase in mutant frequency
was observed (Litton Bionetics, 1982a).
CHO/HGPRT forward mutation assay
TBHQ was not active in the CHO/HGPRT forward mutation assay at
dose levels up to 6 ng/ml without metabolic activation and up to
0.25 mg/ml with S-9 activation (Beilman & Barber, 1985).
Mouse bone marrow cytogenetic assay
In the bone marrow cytogenetic assay, chromosomal aberrations
were not observed in the bone marrow cells of male mice dosed with
TBHQ at levels up to 200 mg/kg b.w. In this study, the sampling time
was 24 hours post-treatment. However, the validity of this study is
questionable, because it is necessary to obtain an earlier sample
(6-12 hours) to detect potential damage (Litton Bionetics, 1985).
Micronuclei formation in mouse bone marrow
Groups of mice were dosed orally on 2 successive days with TBHQ
at doses of 162, 325, or 650 mg/kg. Control groups were treated with
the solvent, dimethylsulfoxide; triethylenemelamine was used as a
positive control. Six hours after the last treatment the animals were
killed and bone marrow preparations were made. There were no
significant increases in the frequency of micronuclei in the test
groups compared to the solvent control (Litton Bionetics, 1982b).
In another study, Swiss mice (Carworth Farm Lane-Petter) were
dosed orally on 2 successive days with 250 mg/kg TBHQ in corn oil.
Control animals received corn oil alone; positive controls received
benzene. Animals in the test group were killed 24, 48, and 72 hours
after the last treatment (those in the positive control group were
killed 24 hours post-treatment) and bone marrow preparations were
made. For the group treated with TBHQ and killed at 24 hours, there
was a statistically-significant increase in the frequency of
micronuclei (p < 0.01). However, this effect was not observed in the
groups killed at 48 and 72 hours (Société Kemin Europa, 1982b).
Dominant lethal study in male rats
Dominant lethal effects were not reported in a study in which
male rats (CRL;COBS,CDC(SD)BR) were fed dietary levels of TBHQ up to
565 mg/kg b.w./day for 83 days. However, the data are insufficient to
draw a conclusion on the dominant lethality of TBHQ, because of
post-implantation losses and early and late deaths of females at the
lower levels tested and lack of information on the number of females
with one or more dead implants (Krasavage & Faber, 1983).
Chromosome abberations in V-79 cells
TBHQ was tested at doses up to 0.33 mg/ml for its ability to
induce chromosomal abberations in V-79 cells in the absence and
presence of a metabolic activation system (S-9 fraction from Arochlor
1254-induced rat liver). There were statistically-significant
increases in the number of ascentric chromosomes at the 0.33 µg/ml
dose (p < 0.05) and chromatid fragments at 0.33 (p < 0.02) and
1.0 µg/ml without metabolic activation. No statistically-significant
changes were observed with metabolic activation (Société Kemin Europa,
1982c).
Special study on short-term pathological and proliferative effects
of TBHQ in the forestomach of Fischer 344 rats
TBHQ was incorporated into the diet of weanling male Fischer 344
rats at dose levels of 0.25 and 1%. After 9 days on the test diet, and
one hour before sacrifice, the test animals were injected i.p. with
0.25 uCi/g methyl-3H-thymidine. The thymidine-labelling index in the
pre-fundic region of the stomach was determined using a standard
technique. At the high dose there was a significant increase in the
labelling index (p < 0.002). The increase was about 50% of that
obtained with rats treated with 0.5% BHA under similar conditions.
Histopathologically, 1% TBHQ led to hyperplasia of the basal cell
layer in the forestomach epithelium; no changes were observed at the
low dose (Nera et al., 1984).
Comments
Bacterial in vitro, mammalian cell in vitro, and in vivo
mutagenicity studies have been performed. It is unclear from the
results of these studies whether TBHQ has genotoxic activity.
Additional studies are necessary to resolve this issue.
The Committee re-examined the 20-month study in rats that was
reviewed at the nineteenth meeting (Annex 1, reference 39). Although
no adverse effects were noted in this study, it is inadequate as a
test for carcinogenicity by present-day standards because (1) there is
no evidence that the level of TBHQ fed was the maximum tolerated dose,
(2) the duration of the test was inadequate, and (3) there was a loss
of animals during the course of the study. The Committee was informed
that lifetime studies in 2 species will be performed in accordance
with current standards for carcinogenicity testing.
The evaluation was based on the previously-reported long-term
feeding study in dogs in which a no-effect level of 1500 ppm in the
diet was established (Annex 1, reference 39).
EVALUATION
Level causing no toxicological effect in the dog
1500 ppm in the diet, equivalent to 37.5 mg/kg b.w./day.
Estimate of temporary acceptable daily intake for man
0-0.2 mg/kg b.w.
Further work or information
Required (by 1990)
Results of lifetime feeding studies in 2 rodent species. Feeding
studies should take into account the normal degradation products of
TBHQ in food.
Desired
Additional studies to resolve questions related to the
genotoxicity of TBHQ.
REFERENCES
Beilman, J.J. & Barber, E.D. (1985). Evaluation of mono-t-
butylhydroquine in the CHO/HGRPT forward mutation assay.
Unpublished report No. 85-0061 from Health and Environment
Laboratories, Eastman Kodak Co., Rochester, NY, USA. Submitted
to WHO by Eastman Kodak Co., Kingsport, TN, USA.
Krasavage, W.J. & Faber, W.D. (1983). Tertiary butylhydroquinone
(TBHQ): dominant lethal assay in rats. Unpublished report from
Health and Environment Laboratories, Eastman Kodak Co.,
Rochester, NY, USA. Submitted to WHO by Eastman Kodak Co.,
Kingsport, TN, USA.
Krasavage, W.J. (1984). The lack of effect of tertiary butyl-
hydroquinone on prothrombin time in male rats. Drug Chem.
Toxicol., 7, 329-334.
Litton Bionetics (1982a). Mutagenicity evaluation of EK 81-0318 (TBHQ)
in the mouse lymphoma forward mutation assay. Unpublished report
No. 20989 from Litton Bionetics Inc. Submitted to WHO by Eastman
Kodak Co., Kingsport, TN, USA.
Litton Bionetics (1982b). Mutagenicity evaluation of EK 81-0318 (TBHQ)
in the mouse micronucleus test. Unpublished report No. 20996 from
Litton bionetics Inc. Submitted to WHO by Eastman Kodak CO.,
Kingsport, TN, USA.
Litton Bionetics (1985). Mutagenicity evaluation of mono-
tertbutylhydroquine in the mouse bone marrow cytogenetic
assay. Unpublished report No. 22202 from Litton Bionetics Inc.
Submitted to WHO by Eastman Kodak Co., Kingsport, TN, USA.
Mueller, K.R. & Lockhart, B.B. Jr. (1983). In vitro genetic activity
report: evaluation of mono-tertiary butylhydroquinone in the Ames
Salmonella/microsome bacterial mutagenesis test. Unpublished
report from Health, Safety and Human Factors Laboratory, Eastman
Kodak Co., Rochester, NY, USA. Submitted to WHO by Eastman
Kodak Co., Kingsport, TN, USA.
Nera, E.A., Lik, E., Iverson, F., Ormsky, E., Harpinsky, K.F., &
Clayson, D.B. (1984). Short-term proliferative effects of BHA and
other phenolic antioxidants in the forestomach of Fischer 344
rats. Toxicol., 32, 197-213.
Société Kemin Europa (1982a). Recherche de mutagénicité sur
Salmonella typhimurium BIS selon la technique de B.N. Ames sur
le product TBHQ. Unpublished report No. 1PL-R-82044 prepared for
Société Kemin Europa, S.A.
Société Kemin Europa (1982b). Recherche de l'éventuelle potentialité
mutagène de la tertio-butylhydroquinone par la technique du
micronucleus chez la souris, C.E.R.T.I. Unpublished report
No. 678 prepared for Société Kemin Europa, S.A.
Société Kemin Europa (1982c). Recherche d'aberrations chromosomiques
par analyse de métaphases sur cellules V79, produit TBHQ,
Institut Pasteur de Lille. Unpublished report No. IPL-R-82050
prepared for Société Kemin Europa, S.A.