Richard A. Ford

Correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern.

The relationship between chemical structure and toxicity was explored through the compilation of a large reference database consisting of over 600 chemical substances tested for a variety of endpoints resulting in over 2900 no-observed-effect levels (NOELs). Each substance in the database was classified into one of three structural classes using a decision tree approach. The resulting cumulative distributions of NOELs for each of the structural classes differed significantly from one another, supporting the contention that chemical structure defines toxicity. The database was used to derive a threshold of acceptable human exposure for each of the structural classes that could be applied in the absence of specific toxicity data on a substance within one of the three structural classes. The human exposure thresholds provide guidance on the degree of testing and evaluation required for substances that lack toxicity data.

Environmental risk assessment for the polycyclic musks AHTN and HHCB in the EU: I. Fate and exposure assessment

For the environmental exposure assessment of the fragrance ingredients 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB) the following properties were determined: vapour pressure 0.0682 and 0.0727 Pa; water solubility 1.25 and 1.75 mg/l; log K(ow) 5.7 and 5.9; log K(oc) 4.80 and 4.86; bioconcentration factor in fish: 597 and 1584 (fresh weight) for AHTN and HHCB, respectively. Both substances are degraded to more polar metabolites in fish, in soil and during sewage treatment. A review is made of concentrations measured in sludge, in freshwater and marine systems including suspended matter, sediment and fish. The 90th-percentile in more than 200 surface water samples is 0.3 microg/l for AHTN and 0.5 microg/l for HHCB. The 90th-percentile of the concentrations in fish is 0.12 mg/kg fresh weight for both substances (n = 27). These concentrations are lower by a factor of 5-15 than predicted on the basis of the yearly use volumes in Europe, 585 tonnes for AHTN and 1482 tonnes for HHCB. Concentrations in sludge-amended soils and in earthworms are predicted based on concentrations measured in sludge. For AHTN, the predicted values are: PECsoil, 0.029 mg/kg and PECworm, 0.065 mg/kg while for HHCB the corresponding figures are 0.032 and 0.099 mg/kg. These concentrations assume a biodegradation half-life in the soil of 180 days based on preliminary soil biodegradation data.

Environmental risk assessment for the polycyclic musks, AHTN and HHCB : II. Effect assessment and risk characterisation

Reports of the polycyclic musks AHTN and HHCB in surface water and fish, primarily in Europe, have prompted studies of their environmental effects. These effects then are used, along with the predicted environmental concentrations in a risk assessment according to the approach developed under European Union Regulation 793/93, in line with the Technical Guidance Document for risk assessment of new and existing chemicals. In 72-h studies with algae (Pseudokirchneriella subcapitata), NOECs were 0.374 mg/l (AHTN) and 0.201 mg/l (HHCB). In 21-day reproductive tests with daphnia (Daphnia magna) NOECs were 0.196 (AHTN) and 0.111 mg/l (HHCB). In 21-day growth tests with bluegill sunfish (Lepomis macrochirus), NOECs were 0.067 (AHTN) and 0.068 mg/l (HHCB). And, finally 35-day early life stage tests with fathead minnows (Pimephales promelas) resulted in NOECs of 0.035 (AHTN) and 0.068 mg/l (HHCB). These results lead to Predicted No Effect Concentrations (PNEC) of 3.5 microg/l (AHTN) and 6.8 microg/l (HHCB) for aquatic organisms. For the soil compartment, 8-week studies with earthworms (Eisenia fetida) resulted in NOECs of 105 (AHTN) and 45 mg/kg (HHCB) and 4-week studies with springtails (Folsomia candida) resulted in a NOECs of 45 mg/kg for both substances. These values lead to a PNEC of 0.32 mg/kg dw for both materials. Using mammalian studies, PNECs for fish or worm eating predators of 10 mg/kg fw (AHTN) and 100 mg/kg fw (HHCB) can be derived. For sediment dwelling organisms, PNECs were derived by equilibrium partitioning using the aquatic PNECs. Comparing PNECs with the measured or predicted environmental exposures leads to risk characterisation ratios as follows: aquatic species: AHTN 0.086, HHCB 0.074; sediment organisms: AHTN 0.44, HHCB 0.064; soil organisms: AHTN 0.091, HHCB 0.10; fish eating predators: AHTN 0.012, HHCB 0.001; worm eating predators: AHTN 0.007, HHCB 0.001.

Comparative metabolism and kinetics of coumarin in mice and rats

Coumarin, a well recognized rat hepatotoxicant, also causes acute, selective necrosis of terminal bronchiolar Clara cells in the mouse lung. Further, chronic oral gavage administration of coumarin at 200 mg/kg, a dose that causes Clara cell death, resulted in a statistically significant increased incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. In contrast, mouse lung tumors were not observed at the 100 and 50 mg/kg dose levels in the oral gavage study, or in CD-1 mice following chronic intake of coumarin at levels equivalent to 276 mg/kg in diet. The current studies were designed to determine the impact of oral gavage vs dietary administration on the pharmacokinetics and metabolism of coumarin in CD-1 and B6C3F1 mice and F344 rats. Following the administration of 200 mg/kg 14C-coumarin via oral gavage, lung C(max) values (total 14C-associated radioactivity) were five- and 37-fold greater than those resulting from a 50 mg/kg oral gavage dose or 1000 ppm in diet, respectively. Coumarin (200 mg/kg) pharmacokinetics and metabolism was also examined in F344 rats following oral gavage dosing. Total 14C-coumarin associated radioactivity in plasma was 3.5-fold lower than in the mouse, and the plasma half-life in rats was five-times longer than in mice. Using non-radiolabeled compound (200 mg/kg), coumarin and products of the coumarin 3,4-epoxidation pathway were quantitated in plasma and urine after oral gavage administration to mice and rats. 7-Hydroxycoumarin (7-HC) was quantitated in mouse plasma and urine. o-Hydroxyphenylacetic acid (o-HPAA) reached a concentration of 37 microg/ml in plasma, and accounted for 41% of the dose in the urine, whereas the C(max) for 7-hydroxycoumarin was 3 microg/ml, and represented 7% of the administered dose. In the rat, the plasma C(max) for o-HPAA was 6 microg/ml, and accounted for 12% of the dose. The coumarin C(max) in rat plasma was comparable to that in mouse. Coumarin 3,4-epoxide (CE) and its rearrangement product o-hydroxyphenylacetaldehyde (o-HPA) and o-hydroxyphenylethanol (o-HPE), were not detected at any time point in plasma or urine. This analysis of coumarin and CE pharmacokinetics in rodents suggests that the differential tumor response in the mouse oral gavage and dietary bioassays is a function of the route of exposure, whereas species differences in lung toxicity between mice and rats result from heightened local bioactivation in the mouse lung.

Neurotoxic properties of musk ambrette

Musk ambrette (2,6-dinitro-3-methoxy-4-tert-butyltoluene), a nitro-musk compound widely used as a fixative in fragrance formulations and found to a lesser degree in flavor compositions, produces hindlimb weakness when administered in the diet or applied to skin of rats for periods up to 12 weeks. Underlying neuropathologic changes consist of primary demyelination and distal axonal degeneration in selected regions of the central and peripheral nervous system. Murine neurological disease induced by musk ambrette occurs at doses well above estimated maximum daily human exposure. Lifetime experimental neurotoxicology studies using lower concentrations of musk ambrette for prolonged periods would be needed for the estimation of human risk.

The in vivo dermal absorption and metabolism of [4-14C] coumarin by rats and by human volunteers under simulated conditions of use in fragrances.

The disposition and metabolic fate of [4-14C]coumarin in a 70% aqueous ethanol solution was studied in male Lister Hooded rats after occluded dermal application and in three male volunteers after an exposure designed to simulate that which may be encountered when using an alcohol-based perfumed product. In both cases, the 6-h exposure was 0.02 mg/cm(2) (rats 0.023 mg/kg and humans 0.77 mg/kg). In both, coumarin was quickly absorbed, distributed and excreted in urine and feces, although fecal excretion of coumarin in humans was only 1% of the applied dose as opposed to 21% in rats. Total absorption was 72% of the applied dose with rats and 60% with humans. Peak plasma radioactivity in both was at 1 h. The mean plasma half-life of coumarin and metabolites was approximately 1.7 h for humans and 5 h for rats. In humans, coumarin was primarily metabolized to and excreted in urine as 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulfate. Small amounts of unconjugated 7-hydroxycoumarin and o-hydroxyphenylacetic acid (o-HPAA) were also excreted. In rats, about twenty metabolites were present, but only o-HPAA was identified. These studies show the rat is a very poor model for humans and toxicity in the rat cannot be extrapolated to humans.

Dermal absorption and disposition of musk ambrette, musk ketone and musk xylene in human subjects.

Musk ambrette, musk ketone and musk xylene have a long history of use as fragrance ingredients, although musk ambrette is no longer used in fragrances. As part of the review of the safety of these uses, it is important to consider the systemic exposure that results from these uses. Since the primary route of exposure to fragrances is on the skin, dermal doses of carbon-14 labelled musk ambrette, musk ketone and musk xylene were applied to the backs (100 cm2) of healthy human volunteers (two to three subjects) at a nominal dose level of 10-20 microg/cm2 and excess material removed at 6 h. Means of 2.0% musk ambrette, 0.5% musk ketone and 0.3% musk xylene were absorbed based on the amounts excreted in urine and faeces during 5 days. Most of the material was excreted in the urine with less than 10% of the amount excreted being found in faeces. No radioactivity was detected in any plasma sample, consistent with low absorption, and no radioactivity was detected (<0.02% dose) in skin strips taken at 120 h. Analysis of urine samples indicated that all three compounds were excreted mainly as single glucuronide conjugates. The aglycones were chromatographically different, but of similar polarity, to the major rat metabolites excreted in bile also as glucuronides.

Evaluation of the oral subchronic toxicity of HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-γ-2-benzopyran) in the rat

1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB) is used in a wide variety of fragrances in consumer products. Because of the widespread exposure to this material, a 90-day oral feeding study in accordance with OECD guidelines No. 408, with 4-week recovery periods for selected rats, was conducted. HHCB was added to the diet of rats at levels calculated to result in mean daily doses of 5, 15, 50 or 150 mg HHCB/kg. On completion of the treatment period, three males and three females from the high dose and control groups were maintained for a treatment free period of 4 weeks. No adverse effects were revealed from clinical examination or following extensive histopathological examinations. There were no significant findings at any dose level; a NOAEL of 150 mg/kg per day was concluded. As a supplement to the main study, histopathological examination of the prostate, seminal vesicles, mammary gland and testes of males and ovaries, mammary gland, uterus and vagina of females was undertaken on all animals in all test groups. This examination did not reveal any evidence of hormonal effects.

The Toxicology and Safety of Fragrances

The consumer today hears more about toxicology and safety than even health professionals did 20 years ago. Yet it is the rare consumer that purchases a fine perfume, an aftershave, a cosmetic or any other of the hundreds of available fragranced products and questions whether it is safe to use. There are several reasons for this.

Dermal absorption and disposition of musk ambrette, musk ketone and musk xylene in rats

Dermal doses of carbon-14 labelled musk ambrette (MA), musk ketone (MK) or musk xylene (MX) to male Sprague-Dawley CD rats were applied at a nominal dose level of 0.5 mg/kg (11 microg/cm2 of skin) and excess material removed at 6 h. Means of about 40, 31 and 19% of the applied doses of MA, MK and MX, respectively, were absorbed. Most of the absorbed material was excreted within 5 days with only 1-2% of the applied dose remaining in the animal at this time. Tissue concentrations of radiolabel were similar for all three compounds with peak concentrations occurring at 6-8 h. In general, fat and liver contained the highest concentrations at around 0.2 microg nitromusk equivalents/g but concentrations in fat declined fairly rapidly to around 0.005 microg equiv./g at 120 h. Most of the absorbed dose was eliminated in bile mainly in the form of polar conjugated metabolites. Structural characterisation of the major aglycones for MA and MX indicated that they were hydroxylated analogues formed by oxidation of the ring methyl. Repeated daily dosing for 14 days resulted in little bioaccumulation for musk xylene and accumulation of about three-fold for musk ketone.