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Description of key information

Oral absorption: 50%
Dermal absorption: 5.2% based on available human dermal absorption study
Inhalation absorption: 100%

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
5.2
Absorption rate - inhalation (%):
100

Additional information

HHCB toxico-kinetic statement 


Introduction


The available toxico-kinetic information in reports and publications is summarised in the EU-RAR (2008) in more detail and this information is attached in the Endpoint summary. In this current toxico-kinetic statement, the key studies are presented from the EU-RAR and information from Api et al. (2013) publication is added who present some new insights based on the studies covered in the EU-RAR (2008).


The test material HHCB (Cas no. 1222-05-5 ) is a polycyclic musk, a hexyl ring with an ether bond, with a benzyl and a pentyl ring attached to it. On the pentyl ring methyl groups are attached to each C atom (Fig. 1). The substance is a highly viscous liquid with a molecular weight of 258.4 that does not preclude absorption. The test material is not likely to hydrolyse and has a low volatility (0.0727 Pa).


Absorption, Oral: The relatively low molecular weight and the moderate octanol/water partition coefficient (Log Kow 5.3 and water solubility (1.65 mg/l) would allow absorption through the gut. Oral absorption is also shown in kinetic studies after oral exposure. The results of the repeated dose and reproductive toxicity studies show that the substance is being absorbed by the gastro-intestinal tract following oral administration based on increased liver and thyroid weights. According to Martinez and Amidon (2002) the optimal log Kow for oral absorption falls within a range of 2-7. This shows that HHCB is likely to be absorbed orally and therefore the oral absorption is expected to be > 50%. 


Skin: Based on the physico-chemical characteristics of the substance, being a liquid, its molecular weight (MW 258.4, log Kow (5.3) and water solubility (1.65 mg/l), indicate that (some) dermal absorption is likely to occur. Several experimental studies are available to determine dermal absorption. The one key study in humans is presented here, all other studies s are presented in the EU-RAR, 2008.


In the key study in human skin, the dermal absorption (non-GLP, but with QA statement) of HHCB was determined over a 24-hr period according to the methodology of the SCCNFP. Radiolabelled HHCB (uniformly labelled in the aromatic ring – radiochemical purity 99.3%) was applied in 1% solution in ethanol (96% v/v) to human epidermal membranes (prepared from female breast or abdominal skin and assayed for integrity with tritiated water) supported on a piece of filter paper (for strength) in glass diffusion cells (n=12). The area of the membrane available for absorption was approximately 1 cm2 and the average applied dose was 20±0.2 µL/cm2. The amount of material absorbed into the receptor phase, 6% Volpo N20 (to enhance solubility) in pH 7.4 phosphate buffered saline, after 24 hr was 0.40±0.06% of the applied dose. The majority of applied HHCB (81±2% of the applied dose) was found in the 24-hr surface wipe and donor chamber wash plus wipe. The stratum corneum tape strips contained 5.8±0.8% of the applied dose and the remaining stratum corneum plus epidermis 4.5±0.6% of the applied dose. Levels of HHCB in the remaining stratum corneum plus epidermis, filter paper (on which the epidermis samples rested) and permeated HHCB were combined to produce a total absorbed dose value of 5.2±0.6% of the applied dose. Overall recovery of radioactivity was 92±0.8% (Green and Brain, 2001).


Lungs: Absorption via the lungs is also indicated based on these physico-chemical properties. Though the inhalation exposure route is thought minor, because of its low volatility (0.0727 Pa), the octanol/water partition coefficient (Log Kow 5.3), indicates that inhalation absorption is possible. The blood/air (BA) partition coefficient is another partition coefficient indicating lung absorption. Buist et al. 2012 have developed BA model for humans using the most important and readily available parameters:


Log PBA = 6.96 – 1.04 Log (VP) – 0.533 (Log) Kow – 0.00495 MW.


For HHCB the B/A partition coefficient would result in:


Log P (BA) = 6.96 – 1.04 Log (0.0727) – 0.533*5.3 – 0.00495 258.4 = 4.04


This means that the substance has a high tendency to go from air into the blood. It should, however, be noted that this regression line is only valid for substances which have a vapour pressure > 100 Pa. Despite HHCB being somewhat out of the applicability domain and the exact B/A may not be fully correct, it can be seen that the substance will be readily absorbed via the inhalation route and will be close to 100%.


Distribution theoretical: The fairly low water solubility of the test substance would limit distribution in the body via the water channels. The log Kow would suggest that the substance would pass through the biological cell membrane. Due to the expected metabolisation the substance as such would show some accumulation in body fat. The experimental distribution information is summarised in the EU-RAR (2008) and in Api et al. (2013). Two studies are presented here, where HHCB is applied via intravenous and oral gavage route.


In an iv study at 2 mg/kg bw HHCB and metabolites were traced in plasma. The data are presented in the following table.


EU-RAR Table 4.9 Concentrations of radioactivity in tissues after an intravenous doses of 14C- HHCB of 2 mg/kg bw to rats (in µg equivalents/g tissue).



























































































































Time



Tissues



Plasma



Whole Blood



Liver



Kidney



Fat



5 min



2.57



1.58



8.83



4.65



1.21



15 min



1.94



1.17



5.90



3.11



1.84



30 min



1.54



0.914



4.73



2.38



3.29



1 hr



1.46



0.845



4.03



1.94



5.27



2 hr



1.31



0.716



3.00



1.26



6.64



4 hr



1.06



0.584



2.32



0.914



5.55



6 hr



1.04



0.565



2.39



0.817



4.20



12 hr



0.564



0.332



1.99



0.503



4.75



24 hr



0.249



0.148



1.09



0.227



3.66



2 days



0.102



0.0644



0.548



0.0920



2.17



7 days



0.011



0.0108



0.121



0.0237



0.575



14 days



0.00199



0.00438



0.0407



0.00985



0.0989



28 days



0.00050



0.00185



0.0221



0.00415



0.0260



 


In an oral study with pregnant and later lactating rats the distribution of orally dosed HHCB and HHCB metabolites was shown in plasma and milk.


Plasma: In the table below the oral doses of HHCB and the concentrations of HHCB and metabolites in plasma are shown. At day 3 and 7 samples were taken after 4, 8 and 24 hours. It can be seen that exposure to 2 mg/kg bw results in almost 2 mg/l HHCB and its metabolites. Increasing the dose to 20 mg/kg bw, the concentrations are 11 mg/l. It can be seen that at both doses and at both sampling days the half-life is < 24 hours.


EU-RAR Table 4.13 Analysis of total radioactivity in plasma after daily oral administration of 2 or 20 mg/kg 14C-HHCB in μg equivalents HHCB/ml plasma
















































Time after parturition



Time after oral administration (hours)



Mean level after oral doses of 2 mg/kg/day



Mean level after oral doses of 20 mg/kg/day



Day 3



4



1.90 ± 0.87



11.08 ± 1.86



 



8



1.05 ± 0.43



7.24 ± 0.53



 



24



0.33 ± 0.12



2.66 ± 0.62



Day 7



4



1.21 ± 0.09



8.76 ± 1.57



 



8



0.66 ± 0.25



5.06 ± 0.70



 



24



0.23 ± 0.05



1.62 ± 0.55



In addition to plasma concentration measures also milk concentrations were measured (EU-RAR Table 4.14 below. It can be seen that HHCB in plasma and milk decreases during sampling time and half-lives of << 24 h are seen.


EU RAR Table 4.14 Analysis of total radioactivity and unchanged HHCB in milk after daily oral administration of 2 or 20 mg/kg 14C-HHCB in μg equivalents HHCB/ml milk (ppm)






















































































 



 



After oral doses of 2 mg/kg bw/day



 



 



After oral doses of 20 mg/kg bw/day



 



 



 



Time after oral dosage (hrs)



Total radiolabel Mean



HHCB


Mean



Ratio


HHCB/total residue



Total radiolabel Mean



HHCB


Mean



Ratio


HHCB/total residue



Day 3



4



1.71 ± 0.20



0.82 ± 0.11



0.48 ± 0.03



32.8 ± 10.9



17.57 ± 6.4



0.53 ± 0.08



 



8



0.88 ± 0.20



0.27 ± 0.09



0.32 ± 0.13



12.4 ± 4.4



4.95 ± 1.48



0.41 ± 0.02



 



24



0.27 ± 0.11



nd



-



1.69 ± 0.37



nd



-



Day 7



4



2.28 ± 0.66



0.99 ± 0.49



0.41 ± 0.11



25.0 ± 7.0



11.56 ± 4.6



0.45 ± 0.06



 



8



1.09 ± 0.20



0.33 ± 0.13



0.30 ± 0.07



16.1 ± 3.55



8.30 ± 2.92



0.52 ± 0.13



 



24



0.15 ± 0.03



nd



-



1.34 ± 0.48



nd



-



nd : not detected due to low radioactivity levels


Metabolism:


Based on the bioaccumulation and degradation data it shows that HHCB is oxidized to a lactone and further de-esterified to a hydroxylated acid as presented below in Fig. 1. This hydroxylated product, which is a secondary alcohol, is likely the handle for glucuronidation as is shown in Api et al. (2013).


 


Fig. 1 HHCB and its metabolites as measured in bioaccumulation and degradation studies and these metabolization steps are also anticipated in rat and pigs. The arrows present the sites for metabolic attack.


 


The Phase 1. Metabolisation of the substance can be deduced based on information from the biodegradation and bioaccumulation studies and the OECD Toolbox 3 rat liver metabolism simulator. The formed metabolites are mainly:


1) the hexylring with the ether bond will be oxidised and form an additional ketone, resulting in an ester (1st metabolite as seen in the biodegradation studies);


2) the hexylring with the ether bond will open and a secondary alcohol can be formed (2nd metabolite, as seen in the biodegradation studies);


3) the methyl groups attached to the pentyl ring can be oxidized into alcohols, aldehydes or acids.


During Phase 2 the formed secondary alcohol (2nd metabolite) will be glucuronidated (Api et al., 2013).


Excretion in faeces and urine: The intravenous and oral studies in rats and the pig showed that HHCB is rapidly distributed. In the rat the excretion is primarily in the faeces. In the pig the principle route of exposure is in the urine. Pig kinetics are more similar to humans than rat and therefore in humans the key route of exposure are the kidneys. In rat the threshold for biliary excretion is 325 Da and substances and conjugated/glucoronidated metabolites exceeding this threshold can be excreted via bile. In pigs and humans the threshold for excretion via bile is higher therefore the glucuronide conjugates of hydroxylated HHCB (MW around 450) exceeds this molecular weight threshold and need to be excreted via the kidneys.


In rats there was no evidence of accumulation, but clearance from the fat was slower than from other tissues as presented in Table 1.


 


Table 1 HHCB concentration in adipose tissue and plasma after exposure of 2 mg/kg bw in rat via iv, after 2, 24 and 178 hours (copied from Api et al.,2013)































Time/hours



HHCB in adipose tissue ug/g



Plasma ug/g



2



3.81



0.094



24



2.45



0.04



168 (7 days)



0.442



Not detectable



Estimated half-life = 50 hours


 



 



See table EU-RAR 4.13 and 4.14 above.



 


Air-breathing organisms, adsorption, distribution, metabolism, excretion and accumulation: Though the bioaccumulation criteria in air breathing organisms are fulfilled for HHCB: Koa > 5 and Kow > 2, other criteria considering absence of metabolism and/or sole excretion via lungs are not fulfilled. The indicative final DT50 cut offs are for final assessment is < 2.5 days for shrews ( a mouse species) or < 70 days in humans (ECHA’s PBT guidance (R.11, page 84).  The HHCB kinetic information shows that the substance is metabolised and is excreted via the kidneys. After dosing of 2 and 20 mg/kg bw the half-lives in plasma and milk are < 24 hours (EU-RAR, 2008, Table 4.13, presented above). In adipose tissue this is ca 50 hours (y=-0.0055x+0.5586 after conversion of the ug/g to log values, as derived from Table 1). This DT50 in adipose rat is < 2.5 days and therefore there is no concern for air-breathing organisms.


Discussion: The substance is expected to be readily absorbed, orally and via inhalation, based on the human and rat toxicological information and physico-chemical parameters. For dermal absorption of HHCB in humans is 5.2% is taken forward to the risk characterization, based on experimental information.


The IGHRC (2006) document of the HSE and mentioned in the ECHA guidance Chapter 8 will be followed to derive the final absorption values for the risk characterisation.


Oral to dermal extrapolation: There are adequate data via the oral route and the critical toxic effect is related to systemic effects and therefore route to route extrapolation is applicable. The toxicity of the substance will be due to the parent compound but also to its metabolites. The overriding principle will be to avoid situations where the extrapolation of data would underestimate toxicity resulting from human exposure to a chemical by the route to route extrapolation. HHCB is not expected to be detoxified in the gut because it is hydrolytically stable. Though some first pass effect via the liver may occur, the toxicity via the dermal route will not be underestimated because absorption will be slower (as has been shown experimentally) and the compound will also pass the liver. Using the asymmetric handling of uncertainty, the oral absorption will be considered 50% (though likely to be higher) and the dermal absorption will be based on the in vitro experimental study: 5.2% for humans.


Oral to inhalation extrapolation: Though HHCB is not a volatile liquid some inhalation exposure will be expected. HHCB is not a corrosive for skin and eye and systemic effects will overrule the effects at the site of contact. In the absence of bioavailability data it is most precautionary that 100% of the inhaled vapour is bioavailable. For inhalation absorption 100% will be used for route to route extrapolation, because this will be precautionary for the inhalation route.


Conclusion: HHCB is expected to be readily absorbed via the oral and inhalation route based on toxicity and physico-chemical data. Using the precautionary principle for route to route extrapolation the final absorption percentages derived are: 50% oral absorption, 5.2% dermal absorption, and 100% inhalation absorption.


References


Api, A-M, Ritacco, G., Sipes, I.G., 2013, Disposition and Excretion of 14C-AHTN (7-Acetyl-1,1,3,4,4,6-Hexamethyl-1,2,3,4-Tetrahydronaphthalene) and 14C-HHCB (1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-Hexamethyl-Cyclopenta-Gamma-2-Benzopyran) After Intravenous Administration to Sprague-Dawley Rats and Domestic Pigs, International Journal of Toxicology, 32, 288-295


 


Buist, H. E., Wit-Bos de, L., Bouwman, T., Vaes, W. H. J., 2012, Predicting blood: air partition coefficient using basis physico-chemical properties, Regul. Toxicol. Pharmacol., 62, 23-28. 


 


EU-RAR, 2008, European Union Risk Assessment Report 1,3,4,6,7,8-HEXAHYDRO-4,6,6,7,8,8-HEXAMETHYLCYCLOPENTA-γ-2- BENZOPYRAN (1,3,4,6,7,8-HEXAHYDRO-4,6,6,7,8,8-HEXAMETHYLIN-DENO[5,6- C]PYRAN - HHCB) CAS No: 1222-05-5 EINECS No: 214-946-9 RISK ASSESSMENT, https://echa.europa.eu/documents/10162/947def3b-bbbf-473b-bc19-3bda7a8da910


 


Martinez, M. N., And Amidon, G. L., 2002, Mechanistic approach to understanding the factors affecting drug absorption: a review of fundament, J. Clinical Pharmacol., 42, 620-643.


 


IGHRC, 2006, Guidelines on route to route extrapolation of toxicity data when assessing health risks of chemicals, http: //ieh. cranfield. ac. uk/ighrc/cr12[1]. pdf