Levels Of Butylated Hydroxytoluene Research Articles

Assessment of Safety, Effects, and Muscle-Specific Accumulation of Dietary Butylated Hydroxytoluene (BHT) in Paralichthys olivaceus.

Butylated hydroxytoluene (BHT) is a commonly used antioxidant added to animal/fish feed to limit lipid autoxidation and peroxidation. Although there have been reviews and reports of BHT toxicity in animals, limited information is available with respect to the toxic effects and accumulation of BHT due to oral exposure in aquaculture species. Therefore, 120 days of feeding trial was conducted to evaluate the effects of dietary BHT on the marine fish olive flounder Paralichthys olivaceus. Graded levels of BHT were added to the basal diet in increments of 0, 10, 20, 40, 80, and 160 mg BHT/kg, corresponding to 0 (BHT0), 11 (BHT11), 19 (BHT19), 35 (BHT35), 85 (BHT85), and 121 (BHT121) mg BHT/kg diets, respectively. Fish with an average weight of 77.5 ± 0.3 g (mean ± SD) were fed one of the six experimental diets in triplicate groups. Growth performance, feed utilization, and survival rate were not significantly affected by the dietary BHT levels among all experimental groups, whereas BHT concentration in the muscle tissue was found to increase in a dose-dependent manner up to 60 days of the experimental period. Thereafter, BHT accumulation in muscle tissue showed a declining trend among all treatment groups. Furthermore, the whole-body proximate composition, nonspecific immune responses, and hematological parameters (except triglycerides) were not significantly influenced by the dietary levels of BHT. Blood triglyceride content was significantly higher in fish fed the BHT-free diet compared to all other treatment groups. Thus, this study demonstrates that dietary BHT (up to 121 mg/kg) is a safe and effective antioxidant without exhibiting any adverse effects on the growth performance, body composition, and immune responses in the marine fish olive flounder, P. olivaceus.

Effects of dietary astaxanthin supplementation on the oxidative stability of meat from suckling lambs fed a commercial milk-replacer containing butylated hydroxytoluene

Meat colour and lipid oxidative stability can be improved by adding antioxidants to animal diet. This study investigated the effects of the addition of astaxanthin to a butylated hydroxytoluene (BHT)-containing commercial milk-replacer, at a rate of 25 mg of astaxanthin/kg of milk-replacer powder, on suckling lamb meat quality. Twenty newborn (2 day old) lambs allocated to individual pens were artificially reared for 22 days. Ten lambs (Control) were fed a commercial milk-replacer and the other ten (Astaxanthin) received the same milk-replacer but included astaxanthin. After the feeding trial, meat and fat colour, astaxanthin and BHT levels in meat, oxidative stability in refrigerated and frozen raw meat and refrigerated cooked meat, and meat volatiles in cooked meat were determined. Astaxanthin in artificially reared suckling lambs at the levels used reduced the accumulation of BHT in the meat, slightly affected meat colour, by reducing meat lightness and increasing meat and fat redness, and increased the lipid stability of frozen meat.

Dietary butylated hydroxytoluene improves lipid metabolism, antioxidant and anti-apoptotic response of largemouth bass (Micropterus salmoides)

A 10-week growth trail was conducted to investigate the efficacy and tolerance of dietary butylated hydroxytoluene (BHT) by evaluating inflammation, apoptosis and hepatic disease related to oxidative stress in largemouth bass (Micropterus salmoides). Four experimental diets were prepared with BHT supplement levels of 0 (B0), 150 (B150), 300 (B300) and 1500 (B1500) mg/kg, in which B150 was at the maximum recommended level established by European Union Regulation, and the B300 and B1500 levels were 2 and 10-fold of B150, respectively. Each diet was fed to 6 replicates with 30 largemouth bass (initial body weight, IBW = 6.20 ± 0.01 g) in each tank. The BHT inclusion level did not affect the specific growth rate, but fish in the B150 group showed the lowest feed conversion rate (P < 0.05). BHT inclusion significantly decreased the levels of plasma TC, TG, LDL, ALT and AKP, and increased the (HDL-C)/TC ratio (P < 0.05). Plasma MDA was significantly decreased in the B150 group and GSH-Px was extremely enhanced in each BHT inclusion group (P < 0.05). Hepatic T-AOC was significantly enhanced and O2− was significantly decreased in each BHT inclusion group compared to the B0 group (P < 0.05), as well as hepatic MDA was significantly decreased in B1500 group (P < 0.05). Dietary BHT inclusion down-regulated the hepatic mRNA levels of inflammation, apoptosis and fibrosis related genes, including TNFα, TGF-β1, α-SMA, IL8, IL11β and caspase-9. Moreover, BHT could improve hepatic lipid metabolism via up-regulating the mRNA levels of APOA1, CYP7A1, CYP8B1, and down-regulating the mRNA levels of PPAR-γ and APOB. Histological examination of the liver morphology with H&E and Sirius Red staining showed that BHT inclusion decreased necrotic degenerative changes and collagen deposition in largemouth bass. An immunofluorescence examination revealed significantly decreased cleaved caspase-3 signals in the BHT groups. In conclusion, the results demonstrated that ROS induces hepatic cell apoptosis and fibrosis via the intrinsic pathway of apoptosis by activating caspase-9 in the mitochondria and then initiates apoptosis by activating caspase-3. Consuming 2.32–23.80 mg/kg·bw/d (150–1500 mg/kg in diet) of BHT effectively improved the plasma and hepatic lipid metabolism, antioxidant response as well as reduced ROS production, protecting hepatic cells from injury. It is implied that even a 10-fold increase of the maximum level of BHT (150 mg/kg) is safe for the largemouth bass.

Effects of Citric Acid and Butylated Hydroxytoluene Alone or In Combination on Prevention of Rancidity of Rice Bran during Storage

Rice bran (RB), a byproduct of rice milling process is a valuable feed resource for the livestock industry. Both lipolytic and oxidative rancidity starts right after bran is removed from the grain during the milling process thereby making long term storage of RB difficult. Anti oxidants such as butylated hydroxytoluene (BHT) control the oxidative rancidity only for a short period of time. Therefore, effective rancidity control measures need to be adopted to maintain the nutritive and feeding value of RB. The objective of this study was to evaluate whether rancidity of RB could be curtailed by storing RB with citric acid (CA) and BHT alone or in combination. RB was collected immediately after milling and stored with three levels of CA (0, 2, and 4%) and three levels of BHT (0, 200, and 400 ppm) in a 3×3 factorial arrangement. Each treatment combination had three replicates. Treated RB was stored in plastic containers with a closed lid for 82 days and analyzed for peroxide value (PV) and pH on 7, 25, 55, 82 days. The mean PV of un-treated RB on day 7 (2.85 meq kg-1) increased to 14.42meq kg-1 during 82 days of storage. When BHT or CA was not used, a significant (p<0.05) negative correlation was found between PV and pH [PV=(7.3291–pH x 0.112; R2=0.901]. Peroxide values were not significantly (p>0.05) affected by BHT or CA on day 7. The use of 0, 200 or 400 ppm BHT significantly reduced the PV during 25 days of storage time, but not thereafter. The use of 2% or 4% CA significantly (p<0.05) reduced the PV up to day 82. It can be concluded that BHT was effective in reducing rancidity of RB only for about month whereas CA controls both lipolytic and oxidative rancidity for about three months

Effects of Citric Acid and Butylated Hydroxytoluene Alone or In Combination on Prevention of Rancidity of Rice Bran during Storage

Rice bran (RB), a byproduct of rice milling process is a valuable feed resource for the livestock industry. Both lipolytic and oxidative rancidity starts right after bran is removed from the grain during the milling process thereby making long term storage of RB difficult. Anti oxidants such as butylated hydroxytoluene (BHT) control the oxidative rancidity only for a short period of time. Therefore, effective rancidity control measures need to be adopted to maintain the nutritive and feeding value of RB. The objective of this study was to evaluate whether rancidity of RB could be curtailed by storing RB with citric acid (CA) and BHT alone or in combination. RB was collected immediately after milling and stored with three levels of CA (0, 2, and 4%) and three levels of BHT (0, 200, and 400 ppm) in a 3×3 factorial arrangement. Each treatment combination had three replicates. Treated RB was stored in plastic containers with a closed lid for 82 days and analyzed for peroxide value (PV) and pH on 7, 25, 55, 82 days. The mean PV of un-treated RB on day 7 (2.85 meq kg-1) increased to 14.42meq kg-1 during 82 days of storage. When BHT or CA was not used, a significant (p<0.05) negative correlation was found between PV and pH [PV=(7.3291–pH x 0.112; R2=0.901]. Peroxide values were not significantly (p>0.05) affected by BHT or CA on day 7. The use of 0, 200 or 400 ppm BHT significantly reduced the PV during 25 days of storage time, but not thereafter. The use of 2% or 4% CA significantly (p<0.05) reduced the PV up to day 82. It can be concluded that BHT was effective in reducing rancidity of RB only for about month whereas CA controls both lipolytic and oxidative rancidity for about three months

Levels of synthetic antioxidants (ethoxyquin, butylated hydroxytoluene and butylated hydroxyanisole) in fish feed and commercially farmed fish

Several synthetic antioxidants are authorized for use as feed additives in the European Union. Ethoxyquin (EQ) and butylated hydroxytoluene (BHT) are generally added to fish meal and fish oil, respectively, to limit lipid oxidation. The study was conducted to examine the concentrations of EQ, BHT and butylated hydroxyanisole (BHA) in several commercially important species of farmed fish, namely Atlantic salmon, halibut and cod and rainbow trout, as well as concentrations in fish feed. The highest levels of BHT, EQ and BHA were found in farmed Atlantic salmon fillets, and were 7.60, 0.17 and 0.07 mg kg−1, respectively. The lowest concentrations of the synthetic antioxidants found were in cod. The concentration of the oxidation product ethoxyquin dimer (EQDM) was more than ten-fold higher than the concentration of parent EQ in Atlantic salmon halibut and rainbow trout, whereas this dimer was not detected in cod fillets. The theoretical consumer exposure to the synthetic antioxidants EQ, BHA and BHT from the consumption of farmed fish was calculated. The contribution of EQ from a single portion (300 g) of skinned fillets of the different species of farmed fish would contribute at most 15% of the acceptable daily intake (ADI) for a 60 kg adult. The consumption of farmed fish would not contribute measurably to the intake of BHA; however, a 300 g portion of farmed Atlantic salmon would contribute up to 75% of the ADI for BHT.

Effects of Butylated Hydroxytoluene on Freezability of Ram Spermatozoa

The present study was conducted to investigate the effects of butylated hydroxytoluene (BHT) supplementation on diluted, cooled and frozen-thawed ram spermatozoa. After primary evaluation of collected ejaculates, only semen samples with motility of more than 70% and sperm concentration higher than 3x10 3 sperm/ml were used for cryopreservation. The selected semen samples were then pooled and diluted 1:4 with Tris Citrate Fructose Yolk (TCFY) extender supplemented with different concentrations of BHT (0.5, 10, 2.0 and 3.0 mM). As the control, semen was diluted and frozen in the diluent without BHT. Motility, progressive motility, viability, membranes and acrosome integrity were evaluated after dilution (part 1), cooling (part 2) and freezing and thawing (part 3). The results of the first part of the experiment showed that there were no significant difference between treatments in the motility, progressive motility, viability, membranes and acrosome integrity of spermatozoa, but the results with 2.0 mM BHT were slightly better than obtained with other levels of BHT and control extender. Significantly better results (p<0.05) were observed in the second part of the experiment for cooled spermatozoa characteristics, when extender was supplemented with 2.0 and 3.0 mM BHT. Furthermore, the results obtained in the third part of the experiment indicated that, after freezing and thawing, all evaluated semen characteristics were improved significantly (p<0.05) by increasing BHT levels, with the best results obtained for extender containing 2 mM BHT. Comparison of these results with those of control diluent, the effects of supplementation were significantly (p<0.01) better. However, the higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of spermatozoa compared to extender containing 2.0 mM BHT. In conclusion, the results obtained in this study showed that the semen quality of rams was improved when BHT was added to extender used before the freezing process.

Residue levels of food-grade antioxidants in postharvest treated in-pod peanuts during five months of storage

In order to investigate residue levels of butylated hydroxyanisole (BHA), propyl paraben (PP) and butylated hydroxytoluene (BHT) during storage, eight-hundred kilograms of bulk peanuts were treated with the following antioxidant emulsions: BHA (1802 μg g −1), BHA–PP (1802 μg g −1 + 1802 μg g −1) M1 and BHA–PP–BHT mixtures (1802 μg g −1 + 901 μg g −1 + 2204 μg g −1) M2 and (1802 μg g −1 + 1802 μg g −1 + 2204 μg g −1) M3. Residues were determined in peanut pod and seed tissues at 1-month intervals during the storage. While the reduction levels of BHA and PP in pods at the end of the storage period ranged from 66% to 76%, BHT levels were decreased extensively (86%). Twenty-four hours after peanuts were treated, antioxidant emulsions effectively seeped into the seeds and low levels of these chemicals were detected during the assay. Residues of PP in seeds were lower (62%) than the other antioxidants. Although the doses used were higher than those approved for food-grade antioxidants in stored peanuts, the residue levels in seeds (32.8–0.02 μg g −1) did not exceed the maximum residue limits during the storage period.

On the anti-atherogenic effect of the antioxidant BHT in cholesterol-fed rabbits: inverse relation between serum triglycerides and atheromatous lesions

We have shown that inclusion of the antioxidant butylated hydroxytoluene (BHT) in the diet protects against development of atherosclerotic lesions in cholesterol-fed rabbits. In parallel, BHT treatment results in increased plasma triglyceride levels. The present study explores the relationship between the triglyceride-inducing and protective effects of BHT in two different studies. The combined material contains 22 rabbits fed cholesterol and 18 rabbits fed cholesterol in combination with 1% BHT. In the BHT group there was an inverse relationship between triglyceride exposure/cholesterol exposure and extent of lesions with r=0.74 ( P=0.0005). Our results show that increased triglyceride exposure parallels the anti-atherogenic effect of BHT. There was no significant correlation between atheromatosis and serum BHT levels. β-Very low density lipoprotein (β-VLDL) from cholesterol and BHT animals was triglyceride-enriched and smaller compared to β-VLDL from cholesterol-fed animals, but there was no significant association between the anti-atherogenic effect of BHT and particle size or apolipoprotein pattern of LDL or β-VLDL. LDL isolated from rabbits treated with cholesterol and BHT was less sensitive to oxidative modification than LDL isolated from rabbits treated with cholesterol only. In conclusion, our results demonstrate that the degree of triglyceride exposure may be an important modulator of the anti-atherogenic effect of an antioxidant.

Three generation toxicity study of butylated hydroxytoluene administered to mice

Butylated hydroxytoluene (BHT) was administered to mice, from 5 weeks of age of the F 0 generation to the weaning of the F 2 generation, in the diet at levels of 0 (control), 0.015, 0.045, 0.135, and 0.405%, and some reproductive and neurobehavioural parameters were measured. There was no consistent significant effect on number of litters, litter size, and litter weight at birth for each generation. The body weight of the pups of the 0.015% BHT group was increased at birth and during the lactation period for each generation. In the F 2 generation, the body weights of the pups of other treatment groups were increased during the lactation period, particularly at postnatal day (PND) 21. In the neurobehavioural parameters, a few parameters were increased in treatment groups; i.e., surface righting at PND 7 in the F 1 generation, surface righting at PND 4 and negative geotaxis at PND 7 in the F 2 generation. The dose levels of BHT in this study showed little adverse effect on reproductive and neurobehavioural parameters on mice.

Disposition of single oral doses of butylated hydroxytoluene in man and rat

The kinetics and metabolism of butylated hydroxytoluene (BHT) in man and rats have been compared. Single oral doses of 200, 63 or 20 mg BHT/kg body weight were administered to rats and a single oral dose of 0.5 mg/kg body weight was ingested by human volunteers (non-smoking males). In rats, kinetic parameters (area under the plasma concentration-time curve, plasma BHT peak levels) showed a dose-dependent increase. Plasma BHT levels after oral administration were about four times higher than those that have been reported for another synthetic food antioxidant, butylated hydroxyanisole (BHA; Verhagen et al., Fd Chem. Toxic. 27, 151–158). This may be a reflection of a smaller volume of distribution for BHT, since there were no differences in plasma elimination half-life or plasma clearance between BHT and BHA. In man, the mean plasma concentration-time profile after oral BHT intake was well below the BHT profiles observed for rats and closely followed plasma BHA kinetics in man. In rats, the simultaneous administration of BHT (200 mg/kg body weight) and BHA (200 mg/kg) significantly decreased the absorption of BHT from the gastro-intestinal tract in the first few hours after treatment; the plasma kinetics of BHA were not influenced by the simultaneous administration of BHT. In human female volunteers no alterations in plasma BHT or BHA profiles were seen after the simultaneous ingestion of BHT (0.25 mg/kg body weight) and BHA (0.25 mg/kg). Rats excrete about 10% of an oral dose of 200 mg BHT/kg as unchanged BHT in the faeces, whereas in man no BHT could be detected in the faeces. Urinary excretion of (un)conjugated 3,5-di- tert-butyl-4-hydroxybenzoic acid (BHT-COOH) accounts for only a small percentage of the administered dose in both rats and humans. It is concluded that the plasma BHT concentrations reached after the administration of a single medium to high dose of BHT to rats or a single low dose to man are very different.

Decrease in blood coagulation factors II (prothrombin), VII, IX and X in the rat after a single oral dose of butylated hydroxytoluene

Male Sprague-Dawley rats were given butylated hydroxytoluene (BHT) in a dose of 800 mg/kg body weight orally, and 0.5–72 hr later plasma concentrations of factors II, VII, IX and X and hepatic levels of BHT and BHT quinone methide (2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone) were determined. Levels of factors II, VII, X and IX were reduced 36–60 hr after BHT treatment, but by 72 hr, those most affected (VII and IX) showed some recovery and X had returned to normal. Hepatic levels of BHT reached a maximum 3 hr (a major peak) and 24 hr after BHT dosing and BHT quinone methide reached a maximum at 6 and 24 hr (a major peak). In rats given BHT orally in doses of 200, 400 and 800 mg/kg, factors II, VII and X decreased after 48 hr only in rats given the highest dosage, but factor IX was more susceptible to BHT and showed a dose-dependent decrease. Phylloquinone (1 mg/rat) injected ip 24 hr after the administration of 800 mg BHT/kg maintained normal levels of factors VII and X and an almost normal level of factor IX, but had little effect on the level of factor II. In studies of the effects of drug-metabolizing-enzyme modifiers, neither ip pretreatment with 75 mg phenobarbital sodium/kg for 3 days nor the feeding of 1% cysteine in the diet throughout the experiment prevented the decrease in vitamin-K-dependent factors by 800 mg BHT/kg, but 2-day ip pretreatment with 60 mg cobaltous chloride/kg/day maintained normal levels of factors II and VII and reduced the BHT effect on factors IX and X. SKF 525A (50 mg/kg) injected ip either 30 min before or 12 hr after BHT treatment partially prevented the decrease in factors II, VII and X, or in all four factors, respectively. Thus the decrease in vitamin K-dependent factors may be the same with a single oral dose of BHT as with dietary BHT, and the anticoagulant effect may require the metabolic activation of BHT.

Inhibition of chemically induced mammary carcinogenesis in rats by long-term exposure to butylated hydroxytoluene (BHT): interrelations among BHT concentration, carcinogen dose, and diet.

In outbred female Sprague-Dawley rats long-term exposure to dietary butylated hydroxytoluene [3,5-di-tert-butyl-4-hydroxytoluene (BHT); CAS: 128-37-0] 1 week before carcinogen administration to termination resulted in a dose-related inhibition of mammary tumorigenesis and adrenocortical nodulogenesis. In animals fed the cereal-based NIH-07 diet and receiving a low dose (5 mg/rat) of 7,12-dimethylbenz [a] anthracene [(DBMA) CAS: 57-97-6], there was a significant overall inhibitory trend in tumor incidence observed among those receiving 300, 1,000, 3,000, and 6,000 ppm BHT. Maximal inhibition was approximately 50% at the highest concentration of BHT (6,000 ppm). The inhibitory effect of BHT on mammary tumor incidence was less pronounced when BHT was administered to rats initiated with a high carcinogen dose: At 15 mg DMBA/rat maximal inhibition was only 20% at the highest concentration of BHT (6,000 ppm). In contrast, when tumor yield was assessed in terms of latency or tumor multiplicity, the inhibitory effect of BHT was more pronounced in the groups given a high dose of DMBA than in the groups given a low dose. In animals given a low dose of DMBA (5 mg) and fed 6,000 ppm BHT in the casein-based AIN-76A diet, tumor incidence was inhibited by 50% of that of the controls; in contrast, when initiation was with a high dose of DMBA (15 mg), tumor incidence was decreased by only 28% of that of the controls. In animals fed the NIH-07 diet, DMBA-induced adrenocortical nodule formation was also inhibited in a dose-dependent fashion by BHT. At 5 mg DMBA maximal inhibition was 86% of control levels (6,000 ppm BHT); at 15 mg DMBA maximal inhibition was 66% of control levels (6,000 ppm BHT). However, when BHT was incorporated into the AIN-76A diet, its inhibitory effects on adrenocortical nodulogenesis were unexpectedly feeble and unrelated to carcinogen dose: In animals initiated with 5 mg DMBA and administered 6,000 ppm BHT, nodule incidence was decreased by only 25%, whereas in animals initiated with 15 mg DMBA, nodule incidence was decreased by 30% of that of the controls. These results indicate that while chronic exposure to dietary BHT suppressed the development of DMBA-induced mammary tumors and adrenocortical nodules, the degree of suppression depended on the dose of carcinogen administered, the level of BHT in the diet, and the parameter being measured. Diet-dependent differences in BHT action were observed with regard to DMBA-induced adrenocortical nodulogenesis but not with regard to mammary tumorigenesis.

Inhibition of norepinephrine uptake into synaptic vesicles by butylated hydroxytoluene

Butylated hydroxytoluene (BHT), a widely used antioxidant food preservative, has been found to be a potent inhibitor of norepinephrine uptake into synaptic vesicles isolated from rat brain. BHT levels as low as one per 400 phospholipid molecules were sufficient to cause 75% inhibition of norepinephrine accumulation at 30°C. The same levels of BHT which inhibited neurotransmitter transport also caused a significant increase in the fluorescence polarization of the membrane probe 1,6-diphenyl-1,3,5-hexatriene embedded in synaptic vesicle membranes, suggesting that BHT may block transport indirectly by decreasing the transmembrane mobility of a norepinephrine carrier protein.

Effects of low levels of butylated hydroxytoluene on the prothrombin index of male rats

Summary Butylated hydroxytoluene (BHT) was administered in the diet to groups of five male rats at concentrations of 0.0085, 0.017, 0.033, 0.065, 0.13, 0.25 and 0.50% for I or 4 wk. A significant decrease in the prothrombin index was observed at wk 1 in each group of rats fed BHT levels of 0.017% or more and this effect was still apparent at wk 4 in the group given 0.50% BHT.

The BHT content of human adipose tissue

Summary A method for determining butylated hydroxytoluene (BHT) in adipose tissue is described. The concentration of BHT found in the body fat was 0·23 ± 0·15 ppm in 11 residents of the UK and 1·30 ± 0·82 ppm in 12 residents of the USA. These concentrations are of the same order of magnitude as those predicted from the data of Daniel et al. ( Fd Cosmet. Toxicol. 1967, 5 , 475) and are 15–80 times less than those found in rats receiving the nountoward-effect level of BHT (50 mg/kg body weight/day) in their diet.

Progress report on multigeneration reproduction studies in rats fed butylated hydroxytoluene (BHT)

Weanling rats have been fed 20% fat diets containing 0, 300, 1000 or 3000 ppm butylated hydroxytoluene (BHT; 3,5-di- tert-butyl-4-hydroxytoluene). At 100 days of age, 16 females on each dietary level were mated with males on the same dietary level. Fertility, parturition and lactation were examined in the parents, and viability, survival, growth, and behaviour examined in the offspring. Ten days after weaning of the first litter, the females were mated a second time and the same examinations made on parents and offspring. Numerous function and clinical tests were performed on the parents after 28 wk on experimental diets and complete gross and microscopic pathology performed after weaning of the second litter, after 42 wk on test diets. After weaning of the second litter, these offspring were fed the same diets as their parents until 100 days of age and mated in the same procedure. The same observations were made on these animals and 2 litters of offspring. A third generation is still under study. At the 3000 ppm dietary level of BHT, a 10–20% reduction in growth rate of parents and offspring was observed. A 20% elevation of serum cholesterol levels was observed after 28 wk, but no cholesterol elevation after 10 wk. A 10–20% increase in relative liver weight was also observed in animals killed after 42 wk on diet. All other observations at 3000 ppm and all observations at 1000 and 300 ppm were comparable with those of the control group. All criteria of reproduction were normal. No teratogenic effects were detected. By all criteria, in 2 generations of parent rats and their offspring, a “no-effect” level of 1000 ppm of BHT in a high fat diet is established.

The effect of the antioxidants, butylated hydroxy anisole, butylated hydroxy toluene and propyl gallate on growth, liver and serum lipids and serum sodium levels of the rat.

SUMMARYConfirmation of our previous finding that there are greater hazards in the use of butylated hydroxy toluene (BHT) than in that of butylated hydroxy anisole (BHA) as antioxidants for use in human foodstuffs was obtained. BHA and propyl gallate (PG) met one of the requirements for safety as a food additive. A sex difference in the effect of BHT was noted; BHT increased the relative weight of the male adrenal and had a greater deleterious effect on the growth rate of the male than of the female rat. A straight line relationship existed between the dietary level of BHT and the serum cholesterol level. When fed at the level of 0·1 p.c. of the diet, BHA increased the level of total serum cholesterol but no further elevations occurred at higher dosages. There was a relatively greater increase in the amount of serum free cholesterol than of ester cholesterol when BHA or BHT were fed. PG had no effect on the serum cholesterol levels. BHT increased both free and esterified adrenal cholesterol, a greater increase being noted in the latter. Although BHA and BHT produced increases in the size of the male adrenal none of the marked histological changes recorded by other workers using lethal doses of these antioxidants in the rabbit were apparent. The antioxidants had no effect on the concentration in the liver of the total and esterified cholesterol, total lipid or on the composition of the polyunsaturated fatty acids. However, as the liver weight was increased by BHA or BHT the absolute lipid content of the liver was increased.