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In vivo studies

Human adipose tissue studies

In a study (non-GLP) to measure residues of HHCB, two human fat samples (origin not specified) were extracted with hexane and the extracts analysed by selective ion trap gas chromatography (GC)/mass spectrometry (MS) for residues of HHCB. Residues were found in both samples at levels of 145 and 149 μg/kg fat (Eschke et al., 1995b).


In this study (non-GLP), human adipose samples were obtained from 8 females and 6 males in Germany between 1993 and 1995. These samples were extracted with a mixture of water/acetone/petroleum ether and analysed for HHCB residues by GC/mass spectroscopy. HHCB was found in all 14 samples at concentrations ranging from 28 to 189 μg/kg fat (ppb) (mean 82). Although the small number of samples and wide range of data preclude meaningful statistical evaluation, a visual inspection of the data reveals no clear correlation with sex or age (Rimkus & Wolf, 1996).


In a non-GLP study, human fat samples obtained over the years 1983/1984 and –1994 in Switzerland from corpses of 10 females and 5 males (age group 3-100 years) were analysed for residues of HHCB by homogenisation followed by extraction with cyclohexane/ethyl acetate (1:1) and analysed by GC/MS. HHCB was detected in all samples with a range of 12 – 171 μg/kg fat (mean 66 μg/kg) (Müller et al., 1996).


In this study, concentrations of PCNs and polycyclic musks were determined in human adipose tissue from Italy collected during 2005–2006. Polycyclic musks as HHCB a were found in 92% of the human samples. HHCB was found at concentrations ranging from <5 to 1435 (mean: 361) ng/g lw in human adipose tissue (Schiavone et al., 2010).


Human blood studies

Blood samples from 413 German subjects (85 men and 328 women) were extracted (details not reported) and analysed by GC-MS for levels of HHCB as well as for musk ketone and musk xylene for comparison. The average level of HHCB (Galaxolide) was 722 ng/l with a range of <100 (3 samples) to >1600 ng/l (23 samples). The 95-percentile level was 1651 ng/l (Bauer & Frössl, 1999).


Blood samples from 100 Austrian volunteers (55% woman, median age 23, average age 25.5, range from 19 to 43 years) were analysed by GC-MS for the determination of levels of musk fragrance substances. A questionnaire on the use of cosmetics, household products and food were completed by these volunteers. Blood samples were analysed using GC-MES analysis after extensive sample extraction and clean-up. HHCB was not detected in 9% of the samples, with a detection limit of ±30 ng/l. The average level of HHCB found was 594 ng/l with a maximum level of 4100 ng/l. A weak correlation was found between the levels of musk fragrances in blood and the use of cosmetics (Sattelberger et al., 2003; Hutter et al., 2005).


Blood samples from 91 Dutch volunteers (48 males and 43 females, age ranging from 19 till 78 years) were analyzed by GC-MS for the determination of levels of musk fragrance substances after solvent extraction. HHCB was found in all 91 samples. The median level of HHCB was 1.3 ng/g serum with a range of 0.2 to 9.2 ng/g serum. The 95-percentile level was 3.6 ng/g serum (Peters, 2005).

In this study blood serum samples from mother and child (maternal and umbilical cord blood) were tested for presence of a series of chemical compounds. In the study HHCB was found to be the major artificial musk compound in human blood and was present in 38 of the 42 maternal blood samples and in 26 of het 27 cord blood samples. The concentrations of HHCB in maternal blood converted to ng/g lipid, (between 0.15-3.2ng/g for maternal blood, and 0.11-1.6 ng/g for cord blood) were similar to concentrations reported previously for human milk and adipose tissue by Zehringer & Herrmann (2001) and Rimkus & Wolf (1996) (Peters, 2005).


This study examined the concentrations of five nitro musks and six polycyclic musks in blood samples from young healthy volunteers. Blood was taken from 100 healthy students of the Medical University of Vienna. The lipophilic fraction was extracted and after purification analyzed by GC–MS. Study participants also completed a questionnaire on the use of cosmetics, about nutrition and other life-style aspects. Highest percentages of synthetic musks in blood plasma samples were found for galaxolide (91%, median 420 ng L−1). In a multivariate approach only younger age, use of lotion and perfumes did significantly predict blood concentrations of polycyclic musks. For nitro musks except body surface area no significant predictor could be found. High percentage of the population is still exposed to nitro musk compounds although blood concentrations of nitro musks are generally lower than those of polycyclic musks. Compared to earlier investigations performed in the 1990s nitro musks were detected in lower percentages and concentrations. There seems to be no dominant source of nitro musk uptake although relationship to body surface area indicates cosmetic products applied to the skin as the likely origin of plasma concentrations (Hutter et al., 2009).


In this study, concentrations of 11 synthetic musks in women above fifty years were determined and the results were compared with earlier results from samples of young females. Blood was taken from 53 women above 50 years of age, analyzed after an extraction and clean-up step by GC/MS. Tonalide-D3 was used as recovery standard in all samples. Hexachlorobenzene 13C6 was used as internal standard. Study participants also completed a questionnaire on the use of cosmetics, about nutrition and other life-style aspects. Galoxide was detected in higher percentages of the blood plasma samples (89 percent, maximum concentration 6900 ng/L). Regression analysis revealed a significant association of galaxolide concentration with frequent use of perfumes, deodorants and shampoos. From the study group investigated older persons showed higher plasma concentrations. These findings could be due to the higher use of lotions and cremes on face and hands and a more frequent use of skin care products because older persons reported more frequently dry skin (Hutter, et al., 2010).


The human exposure to HHCB and tonalide, AHTN in blood), parabens (urinary parahydroxybenzoic acid, HBA) and triclosan (urinary TCS) – was assessed in 210 Flemish adolescents (14–15 years) and in 204 adults (20–40 years) randomly selected from the general population according to a stratified two stage clustered study design. The aim of this study was to define average levels of exposure in the general Flemish population and to identify determinants of exposure. Average levels (GM (95% CI)) in the Flemish adolescents were 0.717 (0.682–0.753) μg/L for blood HHCB. Inter-individual variability was small for HHCB. All biomarkers were positively associated with the use of PCPs. Additionally, levels of HHCB increased with higher educational level of the adolescents (Den Hond et al., 2013).


Human milk studies

In a study (non-GLP) to determine residues of HHCB, two human milk samples (origin not specified) were extracted with hexane and the extracts analysed by GC/MS for residues of HHCB. Residues were found in both samples at levels of 310 and 360 μg/kg fat or 3.3 and 1.5μg/kg (ppb) whole milk based on measured fat contents of 1.06 and 0.41%, respectively (Eschke et al., 1995b).


In a similar study (non-GLP), five milk samples were obtained from 4 nursing mothers and were extracted according to an AOAC method (Helrich, 1990) and analysed for HHCB residues by selective ion trap GC. All samples contained some HHCB at concentrations ranging from 16 – 108 μg/kg milk fat. The fat contents of these samples were not reported (Rimkus & Wolf, 1996).


In another, larger study (non-GLP) of HHCB residues, milk samples (mean 34 g) were obtained from 107 nursing mothers in Germany (mean age 31.5 years, mean body mass index 24.5 kg/m2 at time of child birth and 23.2 kg/m2 at time of milk sampling) under conditions designed to minimize contamination (all equipment was carefully cleaned and the breast area was cleaned 3 times with cotton wool swabs immersed in propylene glycol). All were asked to report on their use of various household products including soaps, detergents and cosmetics as well as their consumption of fish products. As established in a separate study, the background level of HHCB in cyclohexane extracts of cotton swabs was 0.8 ng/ml before these swabs were used for skin cleaning purposes. After the first, second, third and fourth cleaning steps of breast surface with cotton swabs soaked in propylene glycol, HHCB levels were 127, 182, 136 and 97 ng/ml respectively. Because of the decline after 3 cleanings and in consideration with the mothers, the study authors decided to take milk samples after 3 cleaning steps. HHCB was detected in 82% of all samples. HHCB was detected in the milk samples at levels from zero to maximum 1316 μg/kg of fat with a mean of 80 μg/kg of fat. Based on the reported mean fat level of 3.67 %, this corresponds to a maximum level in the whole milk of 48 μg/kg milk (ppb) with a mean of 2.9 μg/kg milk (ppb). Higher concentrations of HHCB were seen in subjects with a higher body mass index (BMI), either at parturition or the time of milk sampling. This could suggest that an increased fat storage in the body causes an accumulation of synthetic musk fragrances, which in turn leads to a higher concentration of these substances in the breast milk. If this were true, one would find some positive correlations between the BMI change and/or body weight loss versus musk concentrations in breast milk, however, this turns out to be not the case. Another important biological variable is maternal age, which was found to have no bearing on the musk concentrations in milk fat. There was also no correlation shown with number of siblings, complete time of breast feeding, diet or use of household products and cosmetics (Sönnichsen et al., 1999).


In 2001, Zehringer and Herrmann published data on 53 milk samples obtained in 1998/1999 from 29 mothers living around the city of Basle. HHCB was found in concentrations ranging from not-detectable (1 sample) to 281 μg HHCB/kg fat. The average fat content was 3.3%. This resulted in a mean level of 73 μg HHCB/kg fat. No special arrangements were made to prevent contamination of milk samples by HHCB present on the skin (Zehringer & Herrmann, 2001).


In a case control study for risk factors of early miscarriages in the Uppsala County in Sweden, during the period 1996-2003, women donated milk samples to be analysed for the presence of various polycyclic- and nitromusks. Women were also asked to fill out extensive questionnaires about lifestyle, medical history and dietary habits and a sub-population was also asked to fill out an additional questionnaire about use of perfumes and perfumed deodorants, skin lotions, laundry- and washing detergents. Milk was collected at the beginning and at the end of the breast-feeding sessions. The goal was to collect 500 ml from each mother during the 3rd week after delivery. It was not mentioned whether measurements were taken to prevent contamination of the milk samples from musks present on the mother’s skin. In total 101 milk samples were analysed. For 42 of these, useful data on use of perfumed products were available. HHCB was found in concentrations ranging from 2.8 to 268 μg/kg milk (mean ± SD: 78 ± 55; median 64 μg/kg milk fat). No correlation could be demonstrated between concentration of HHCB in milk samples and lifestyle factors, medical history or dietary habits and no time-trend was observed. The results may indicate that concentrations in milk samples from women using perfumed products were higher than in those collected from non-users. However, the differences were only significant for use of perfume during pregnancy (≥ 1/week) as compared to no use, but the significance was driven by only three samples, in the otherwise rather small sample population (Lignell et al., 2004).


In a pilot study in the Czech Republic, 59 milk samples were collected from nursing mothers (living but not necessarily born in Prague). The manual sampling (milk expressed from the breast into a clean container) was conducted in accordance with WHO guidelines. Using a detailed questionnaire, relevant information on parameters, such as age, dietary habits (specifically consumption of freshwater/marine fish), use of perfumed cosmetics, frequency of contacts with detergents, etcetera were collated. The lipid content in the human milk samples ranged from 1.5 to 4.2 wt %. The detection limit for the musks was 10 μg/kg fat. HHCB was found in all the samples. Concentrations ranged from 13 to 720 μg/kg fat, with a median value of 149, a mean value of 214, and a 90th percentile of 509 μg HHCB/kg fat. No correlations were found between the musk levels and personal data of the mothers obtained by the questionnaire (Hajslova & Setkova, 2004).


Milk samples were collected in 1999 at Hvidovre Hospital in Denmark from 10 primiparous mothers (25-29 year of age) 14-26 weeks after birth. HHCB was found at levels from 38.0 to 422 μg/kg fat. Fat content of the milk samples had an average of 3.5% ±2.5% (2.1%-4.8%). The median level of HHCB was 147 μg/kg fat and the average level was 179 μg/kg fat (Duedahl-Olesen et al., 2005).


HHCB, and 4 other polycyclic musks and two nitro musks were analyzed in mother′s milk from 101 primiparae women in Sweden, 1996-2003. Possible temporal trends in musk concentrations and associations with lifestyle/medical factors, such as use of perfumed products during pregnancy were studied. HHCB showed the highest median concentration (63.9 ng/g lipid). Women with a high use of perfume during pregnancy had elevated milk concentrations of HHCB. This strongly suggests that perfumed products are important sources of musk exposure both among the mothers and the nursed infants. No temporal trend in HHCB concentrations was seen (Lignell et al., 2008).


The present study aimed at providing accurate current data on the occurrence of nitro and polycyclic musks in human milk. Samples from 40 healthy breast-feeding mothers were analysed under carefully controlled conditions avoiding secondary contamination. In contrast to the nitro musks, higher contents of polycyclic musk HHCB, were detected here than reported previously by Rimkus &Wolf (1995) (present study mean 115 ng/g fat vs 49 ng/g fat in 1995). Among the polycylic musks HHCB was found in most samples (median 64 ng/kg fat) (Liebl et al., 2000).



In a GLP compliant study, the absorption and excretion of total radioactivity was determined in 3 human male volunteers. 14C- HHCB (uniformly labelled in the aromatic ring – radiochemical purity 98.3%) was applied to the skin of human volunteers under conditions intended to simulate a typically high exposure from the use of alcohol based products such as perfumes or eaux de toilette, i.e. 0.4% in 70% ethanol. A mean of 1.76 mg 14C- HHCB dissolved in 70 % ethanol (0.48 ml) was applied to 100 cm2 (0.018 mg/cm2) area of skin on the upper back. After 30 min to allow the ethanol to evaporate, the area was covered with light gauze dressing. Six hr after application, the dressing was removed and the treated area washed with cotton wool swabs, moistened with 70% ethanol. An area of 6.25 cm2 was stripped by 5 successive applications of adhesive tape to determine the amount of total radioactivity in the upper level of the horny layer. The treated site was again covered with fresh dressings up to 120 hr after compound application at which time the dressings were taken off and another skin area of 6.25 cm2 was stripped to determine the remaining total radioactivity in the upper stratum corneum. Samples of blood (at 0.5, 1, 2, 4. 6, 8, 10, 12, 24, 36, 48, 72, 96 and 120-hr) and excreta (urine at 0-2, 2-4, 4-6, 6-12, 12-24, 24-48, 48-72, 72-96 and 96-120-hr intervals, and faeces at 24 hr intervals) were collected during the five-day period. The majority of the applied material (~56%) was still on the surface of the skin at the time of washing - 6-hr. The first tape stripping at time of removal of the dose indicated that approximately 11% of the applied radioactivity (AR) remained in the upper layers of the stratum corneum. Recovery in the faeces was below the limits of accurate detection (< 0,1 % applied radioactivity, AR) and only one of the three subjects excreted measurable radiolabel in the urine (0.1 % AR). Concentrations in whole blood and plasma were also below the limits of accurate measurement (i.e. < 2.76 ng/ml) at all sampling times. A further 19.5% AR was detected in dressings at the 120 hr time suggesting that considerable radioactivity remained in the skin after washing but was not significantly absorbed. Tape stripping at 120-hr indicated that only trace amounts (0.27% of the dose) remained in the upper layers of the stratum corneum at that time. Assuming that the radioactivity found on the strippings is representative for the entire application site, a total recovery from excreta, dressings, swabs and skin strips of ~ 86.44% AR can be calculated. A separate study indicated that approximately 22% of the HHCB may evaporate under experimental conditions similar to those for this human unoccluded dermal uptake study (Hawkins et al., 1996b; Ford et al., 1999).



-Bauer K and Frössl C (1999). Blutkonzentrationen von polycyclischen und nitromoschusverbindungen bei deutschen probanden. Umwelt medizin gesellschaft 12, 235-237

-Den Hond E, Paulussen M, Geens T, Bruckers L, Baeyens W, David F, Dumont E, Loots I, Morrens B, Nemery de Bellevaux B, Nelen V, Schoeters G, Van Larebeke N, Covaci A (2013). Biomarkers of human exposure to personal care products: Results from the Flemish Environment and Health Study (FLEHS 2007–2011). Science of the Total Environment 463–464 (2013) 102–110

-Duedahl-Olesen L, Cederberg T, Høgsbro Pedersen K and Højgard A (2005). Synthetic musk fragrances in trout from Danish fish farms and human milk. Chemosphere 61, 422-431.

-Eschke HD, Dibowski HJ and Traud J (1995b). Detection and quantitative analysis of musk fragrances by means of ion-trap GC/MS/MS in human fat and breast milk. Deutsche Lebensmittel – Rundschau 91(12), 375-379.

-Ford RA, Hawkins DR, Schwarzenbach R and Api AM (1999). The systemic exposure to the polycyclic musks, AHTN and HHCB, under conditions of use as fragrance ingredients: evidence of lack of complete absorption from a skin reservoir. Toxicology Letters 111, 133-142.

-Hajslova J and Setkova L (2004). Synthetic musks in bioindicators: Monitoring data of fish and human milk samples from the Czech Republic. In: Synthetic musk fragrances in the environment. The Handbook of environmental chemistry (Ed. G.G. Rimkus). Volume 3, Part X, 151-188.

-Hawkins DR, Kirkpatrick D and Girkin R (1995). 14C-HHCB -Studies on the dermal absorption in the rat. Project No. RIF32/951257. Huntingdon Life Sciences Ltd. Report to the Research Institute for Fragrance Materials, Inc. (RIFM).

-Hutter H-P, Wallner P, Moshammer H, Hartl W, Sattelberger R, Lorbeer G and Kundi M (2005). Blood concentrations of polycyclic musks in healthy young adults. Chemosphere 59, 487-492.IFRA (2002).

-Hutter H-P, Wallner P, Moshammer H, Hartl W, Sattelberger R, Lorbeer G and Kundi M (2009). Synthetic musks in blood of healthy young adults: Relationship to cosmetics use. Science of the Total Environment 407 (2009) 4821–4825
-Hutter H-P, Wallner P, Hartl W, Uhl M, Lorbeer G, Gminski R, Mersch-Sundermann V, Kundi M (2010) Higher blood concentrations of synthetic musks in women above fifty years than in younger women. Int. J. Hyg. Environ. Health 213 (2010) 124–130.
-Liebl B, Mayer R, Ommer S, Sönnichsen C., Koletzko B (2000). TRANSITION OF NITRO MUSKS AND POLYCYCLIC MUSKS INTO HUMAN MILK. Short and Long Term Effects of Breast Feeding on Child Health Edited by Berthold Koletzko et al., Kluwer Academic/Plenum Publishers, 2000

-Lignell S, Aune M, Darnerud PO and Glynn A (2004) Synthetic musk components in breastmilk from primiparae women in Uppsala County, Sweden, 1996-2003. Report to the Swedish Environmental Protection Agency, by National Food Administration, Sweden.

-Lignell S, Darnerud PO, Aune M, Cnattingius S, Hajslova J, Setkova L, Glynn A (2008). Temporal Trends of Synthetic Musk Compounds in Mother′s Milk and Associations with Personal Use of Perfumed Products. Environ. Sci. Technol. 2008, 42, 6743–6748

-Müller S, Schmid P and Schlatter C (1996). Occurrence of nitro and non-nitro benzenoid musk compounds in human adipose tissue. Chemosphere 33 (1), 17-28.

-Peters RJB (2004). Man-Made Chemicals in Human Blood. TNO-MEP report R2004/493.

-Peters RJB (2005). Man-Made Chemicals in Maternal and Cord blood. TNO report R2005/129.
-Rimkus GG and Wolf M (1996). Determination of polycyclic musk fragrances in human fat and human milk by GC-EI-MS. Eighteenth International Symposium on Capillary Chromatography, Volume II, Sandra P and Devos G, eds. Palazzo del Congressi, Riva del Garda, Italy, May 20-24, 1996, pages 957-966.

-Sattelberger R, Lorbeer G, Werner H, Hutter H-P and Uhl M (2003). Humanbiomonitoring von moschusduftstoffen. Umwelbundesamt/Federal Environment Agency – Austria.

-Schiavone A, Kannan K, Horii Y, Focardi S, Corsolini S, (2010). Polybrominated diphenyl ethers, polychlorinated naphthalenes and polycyclic musks in human fat from Italy: Comparison to polychlorinated biphenyls and organochlorine pesticides. Environmental Pollution 158 (2010) 599–606

-Sönnichsen C, Mayer R, Ommer S and Koletzko B (1999). Synthetic musk fragrances in human milk. Final report to RIFM. Unpublished report, 3 March.

-Zehringer M and Herrmann A (2001). Analysis of polychlorinated biphenyls, pyrethroid insecticides and fragrances in human milk using a laminar cup liner in the GC injector. Eur. Food Res. Technol. 212, 247-251

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