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.
See Also: Toxicological Abbreviations