Subchronic inhalation studies of complex fragrance mixtures in rats and hamsters.

Users of consumer products are invariably and intentionally exposed to complex mixtures in such products. With finished fragrance products, these mixtures may represent 100 or more fragrance raw materials (FRMs). The objective of the described studies was to evaluate the safety of finished fragrance products via the inhalation route. In total, the finished products contained approximately 100 FRMs at concentrations of 1% or greater. Major FRMs evaluated included benzyl acetate, coumarin, hydroxycitronellal, musk ketone, 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-gamma-2-be nzopyran (HHCB) and phenyl ethyl alcohol. Groups of rats or hamsters were exposed by inhalation (whole body) to the mixtures at 5, 9 or 50 mg/m3 for 4 h per day, 5 days per week for 6 or 13 weeks. For each of the fragrance products, the doses used generally represented a ten- to 100-fold exaggeration of levels expected to be achieved during typical use by consumers. With one exception, the fragrances were aerosolized prior to introduction into the inhalation chamber. The exception product was formulated with a propellant, packaged in a pressurized container and expelled with an automated actuator. In all studies, chamber concentrations of fragrance were monitored. Particle sizes ranged from 0.5 to 7.5 microm, depending on the study. Subchronic exposure to all fragrance mixtures resulted in no toxicologically significant effects on animal survival, behavior, body weights or weight gains, organ weights, or in hematology, clinical chemistry, or urinalysis parameters. No gross pathological or histopathological findings related to test material exposures were observed. These studies support the conclusions that the fragrance mixtures would not pose a hazard to product users based on repeated and exaggerated inhalation exposures of animals.

Reactions of aqueous chlorine and chlorine dioxide with model food compounds.

Chlorine and chlorine dioxide (ClO2), common disinfecting and bleaching chemicals used in the food industry, are potent oxidizing and chlorinating agents. Unfortunately, little is known about the nature of the reactions of chlorine with organic food constituents. This presentation reviews published information concerning the reactions of chlorine gas (Cl2[g]), aqueous chlorine, and ClO2 with model food compounds, the fate of chlorine during the chlorination of specific food products, and the potential toxicity of the reaction products. Fatty acids and their methyl esters react with chlorine with the degree of incorporation corresponding to their degree of unsaturation. Aqueous chlorine oxidizes and chlorinates lipids and amino acids much more readily than ClO2. Several amino acids are highly susceptible to oxidation and chlorination by chlorine compounds. Reactions of chlorine and ClO2 with several food products, including flour and shrimp, have also been characterized. In one model system, 99% of Cl2(g) either reacted with components of flour or was consumed by oxidation/chlorination reactions. The lipids extracted from the chlorinated flour contained significant amounts of chlorine. Exposure of shrimp to hypochlorous acid (HOCl) solution resulted in significant incorporation of chlorine into the edible portion. Although significant quantities of chlorine can be incorporated into specific model compounds and food products, the health risks associated with exposure to chlorinated organic products are unknown. Preliminary studies using the Ames Salmonella/microsome mutagenicity assay indicate that the reaction products from mixtures of aqueous chlorine and various lipids or tryptophan are nonmutagenic. Nevertheless, additional studies are warranted, so that the toxicological significance of these reaction products can be understood more fully.

Production and isolation of aflatoxin M1 for toxicological studies.

One hundred mg aflatoxin M1 was produced and purified for toxicological studies. Aspergillus flavus NRRL 3251 was cultured on rice to produce aflatoxins B1, B2, M1, and M2, B1 and B2 were separated from M1 and M2 by a normal phase low pressure liquid chromatography (LC) column. M1 was then separated from M2 by a reverse phase low pressure LC column. Recoveries of aflatoxins from the LC columns were about 90%. The purified M1 was confirmed by ultraviolet-visible spectrometry, mass spectrometry, nuclear magnetic resonance spectrometry, optical rotation, and its mutagenicity to Salmonella typhimurium TA98.

Effect of butylated hydroxytoluene pretreatment on the excretion, tissue distribution and DNA binding of [14C]aflatoxin B1 in the rat.

The effect of butylated hydroxytoluene (BHT) pretreatment (0.5% in the diet for 10 days) on the excretion, tissue distribution and DNA binding of orally administered [14C]aflatoxin B1 (AFB1) was determined in male Fischer F344 rats. The amount of radioactivity excreted in the urine and faeces by 24 hr was higher in BHT-treated rats than in controls. Treatment with BHT enhanced the excretion of water-soluble metabolites in the urine and in the large intestines plus faeces at the earlier sampling times. The amount of radioactivity bound to hepatic nuclear DNA was six times less in the BHT-pretreated rats than in controls 6 hr after administration of the isotope. The half-lives of [14C]DNA in the rat liver were 30 and 46 hr for control and BHT-pretreated rats, respectively. These results indicate that BHT pretreatment may protect the animal from the carcinogenic effects of AFB1 by enhancing the detoxification and excretion of the mycotoxin.

Effect of butylated hydroxytoluene on the disposition of [14C]aflatoxin B1 in the lactating rat.

The distribution and metabolism of an ip dose of [14C]aflatoxin B1 (AFB1) were studied in lactating Sprague-Dawley rats fed for the previous 13 days on a diet containing 0.5% butylated hydroxytoluene (BHT). Compared with ingestion of a BHT-free diet, treatment with BHT increased the biotransmission of AFB1 metabolites, predominantly aflatoxin M1 (AFM1), into the mammary gland and its content of milk, decreased AFB1 binding to liver nuclear DNA and enhanced the excretion of water-soluble metabolites of AFB1, all measured 6 hr after an oral dose of [14C]AFB1. These changes are related to the induction by BHT of hepatic enzymes involved in the transformation and detoxification of AFB1. The results suggest that exposure to BHT may protect the lactating animal from the carcinogenic effect of AFB1 but may increase the risk of exposure of the newborn infant to the carcinogenic metabolite AFM1.

The effects of butylated hydroxytoluene on the in vitro metabolism, DNA-binding and mutagenicity of aflatoxin B1 in the rat.

Butylated hydroxytoluene pretreatment in the rat enhanced the total in vitro metabolism of aflatoxin B1 by the hepatic postmitochondrial fraction (S-9) and increased the formation of aflatoxin M1, aflatoxin Q1 and a metabolite tentatively identified as the aflatoxin-glutathione conjugate, the latter being the major metabolite produced. Addition of diethyl maleate, a glutathione depletor, to the incubation mix, reduced formation of the conjugate. No significant difference between treated and control animals was observed in the S-9-mediated binding of aflatoxin B1 to calf thymus DNA. However, the mutagenicity of aflatoxin B1 in Salmonella typhimurium TA98 was significantly lower in the presence of S-9 from BHT-treated rats than with S-9 from controls.