Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Endpoint summary

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

There is no reliable and relevant information source in which the toxicokinetic properties (absorption, distribution, metabolism, elimination) of TERPINOLENE MULTICONSTITUENT were investigated. The expected toxicokinetic behaviour is derived from the information available on the main constituents, ie Terpinolene monoconstituent and 1,8-Cineol and are confirmed by the physicochemical properties and the results from the available toxicological data on TERPINOLENE MULTICONSTITUENT following the guide given in the REACH guidance document R.7c.

Information on Terpinolene monoconstituent:

d-limonene and Terpinolene monoconstituent are monocyclic monounsaturated terpenes and they have very similar physic-chemical parameters as shown in the table below, therefore information on the structure-related d-limonene is given because it is considered to be representative of the toxicokinetic properties of Terpinolene monoconstituent.

	Molecular weight	Water solubility (mg/L)	Log Kow	Vapour pressure (Pa at 25°C)
d-limonene	136	Column elution method : 3.99 Slow stirring method : 5.69	4.38	200
Terpinolene monoconstituent	136	Column elution method : 4.55 Slow-stirring method : 7.03	4.33	101

 

Terpinolene monoconstituent is a mono constituent having a relatively low molecular weight of 136. It is a liquid with a low water solubility of about 4.6 mg/L and has high lipophilic properties (log Kow = 4.33). Vapour pressure was determined to be about 101 Pa at 20°C.

Studies of terpene hydrocarbons indicate that they are rapidly absorbed, distributed, metabolized and excreted. The principal metabolic pathway involves side chain oxidation to yield monocyclic terpene alcohols and carboxylic acids. These metabolites are mainly conjugated with glucuronic acid and excreted in the urine, or to a lesser extent in the feces. A secondary pathway involves epoxidation of either the exocyclic or endocyclic double bond yielding an epoxide that is subsequently detoxicated via formation of the corresponding diol or conjugation with glutathione. Humans are continually exposed to limonene and Terpinolene monoconstituent throughout their lifetimes, via consumption of a traditional diet or inhalation of air. Extensive studies on d-limonene show rapid metabolism to polar oxidized metabolites, followed by conjugation and rapid excretion.

 

Absorption:

Terpinolene monoconstituent being lipophilic (log Kow = 4.33), the rate of uptake into the stratum corneum is expected to be high while the rate of penetration is likely to be limited by the rate of transfer between the stratum corneum and the epidermis. Moreover, it is assumed that the dermal uptake is also limited by the low water solubility of Terpinolene monoconstituent. These assumptions are supported by the absence of systemic effects following single-dose dermal application of Terpinolene monoconstituent up to 2000 mg/kg bw which would suggest a limited systemic absorption through cutaneous barriers. Moreover, enhanced skin penetration is not expected since Terpinolene monoconstituent is not a skin irritant or corrosive. However, Terpinolene monoconstituent was found to be skin sensitizing therefore some uptake, even limited, must have occurred. Thus, dermal absorption of Terpinolene monoconstituent is expected to be limited but not inexistent.

In shaved mice, the dermal absorption of [3H]d/l-limonene from bathing water was rapid, reaching the maximum level in 10 minutes (von Schäfer & Schäfer, 1982). In one study (one hand exposed to 98% d-limonene for 2 hours), the dermal uptake of d-limonene in humans was reported to be low compared with that by inhalation (Falk et al., 1991); however, quantitative data were not provided.

 

Terpinolene monoconstituent has high log Kow (>4) and it is a small molecule (molecular weight < 200) therefore, it could be absorbed orally by passive diffusion. It is of adequate molecular size to participate in endogenous absorption mechanisms within the mammalian gastrointestinal tract. Being lipophilic, it may cross gastrointestinal epithelial barriers even if the absorption may be limited by the inability of the substance to dissolve into gastro-intestinal fluids and hence make contact with the mucosal surface. The acute oral gavage toxicity study identified no evidence of systemic toxicity, i.e. neither mortality nor macroscopic effects although high lethal doses were tested (up to 5 mL/kg bw). Oral bioavailability is confirmed in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test where bodyweight gain was reduced at the highest dose group for females. Macroscopic observations at necropsy showed an increase in liver weight both absolute and relative to terminal body weight in males. Histopathology revealed reversible minimal to slight centrilobular hepatocellular hypertrophy in males treated with 2500 and 5000 ppm. These adaptive changes recorded in liver are compatible with metabolism in this detoxifying organ in response to xenobiotic exposure.

Orally administered d-limonene is rapidly and almost completely taken up from the gastrointestinal tract in humans as well as in animals (Igimi et al., 1974; Kodama et al., 1976). Infusion of labelled d-limonene into the common bile duct of volunteers revealed that the chemical was very poorly absorbed from the biliary system (Igimi et al., 1991).

 

Thus, indications of oral uptake of Terpinolene monoconstituent at high doses are given while dermal uptake would be more limited.

 

No study by inhalation was performed. However, considering the low vapour pressure of Terpinolene monoconstituent (<500 Pa), exposure to Terpinolene monoconstituent by inhalation is likely to be very limited.

d-limonene has a high partition coefficient between blood and air and is easily taken up in the blood at the alveolus (Falk et al., 1990). The net uptake of d-limonene in volunteers exposed to the chemical at concentrations of 450, 225, and 10 mg/m3 for 2 hours during light physical exercise averaged 65% (Falk Filipsson et al., 1993).

 

Therefore the potential bioavailability of Terpinolene monoconstituent can be considered mainly by oral route.

Distribution:

Terpinolene monoconstituent is a small molecule with low water solubility and high lipophilicity which indicates that Terpinolene monoconstituent could be widely distributed; based on its lipophilic character, the substance would readily cross cellular barriers or would be distributed into fatty tissues with a low potential to accumulate.

d-Limonene is rapidly distributed to different tissues in the body. A high oil/blood partition coefficient and a long half-life during the slow elimination phase suggest high affinity to adipose tissues (Falk et al., 1990; Falk Filipsson et al., 1993). In rats, the tissue distribution of radioactivity was initially high in the liver, kidneys, and blood after the oral administration of [14C]d-limonene (Igimi et al., 1974); however, negligible amounts of radioactivity were found after 48 hours. Differences between species regarding the renal disposition and protein binding of d-limonene have been observed. For rats, there is also a sex-related variation (Lehman-McKeeman et al., 1989; Webb et al., 1989). The concentration of d-limonene equivalents was about 3 times higher in male rats than in females, and about 40% was reversibly bound to the male rat specific protein, 2µ-globulin (Lehman-McKeeman et al., 1989; Lehman-McKeeman & Caudill, 1992).

Metabolism:

No data are available but, in in vitro genotoxicity studies, differences in cytotoxicity were observed with and without metabolic activation: in Ames test, Terpinolene monoconstituent concentrations leading to cytotoxicity were higher in presence of metabolic activation than without metabolic activation. The same effect was observed in chromosome aberration test on human lymphocytes. This indicates that Terpinolene monoconstituent is metabolised by hepatic microsomal fractions.

Also, in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test, adaptive effects such as increased liver weight and minimal to slight diffuse hepatocellular hypertrophy were observed in males at high doses, which is suggestive of metabolism in this organ.

 

In humans, limonene given orally yields the following major plasma metabolites: perillic acid, limonene-1,2-diol, limonene-8,9-diol, and dihydroperillic acid, probably derived from perillic acid (Poon et al., 1996; Crowell et al., 1994; Vigushin et al., 1998). Peak plasma levels for all metabolites were achieved 4-6 hours after administration, with the exception of limonene-8,9-diol which reached its peak level one hour after administration (Crowell et al., 1994). Phase II glucuronic acid conjugates have been identified in the urine of human volunteers for all metabolites (Poon et al., 1996; Kodama et al., 1974; 1976).

The biotransformation of d-limonene has been studied in many species, with several possible pathways of metabolism (Figure 1). Metabolic differences between species have been observed with respect to the metabolites present in both plasma and urine. About 25–30% of an oral dose of d-limonene in humans was found in urine as d-limonene-8,9-diol and its glucuronide; about 7–11% was eliminated as perillic acid (4-(1- methylethenyl)-1-cyclohexene-1-carboxylic acid) and its metabolites (Smith et al., 1969; Kodama et al., 1976). In another study, perillic acid was reported to be the principal metabolite in plasma in both rats and humans (Crowell et al., 1992). Other reported pathways of limonene metabolism involve ring hydroxylation and oxidation of the methyl group (Kodama et al., 1976). Urinary metabolites isolated from male rabbits orally administered [14C]-d-limonene included perillic acid-8,9-diol (major), p-menth-1,8-dien-10-ol, p-menth-1-ene-8,9-diol, perillic acid, p-mentha-1,8-dien-10-yl glucuronic acid and 8-hydroxy-p-menth-1-en-9-ylbeta-glucopyranosiduronic acid [Kodama et al., 1974].

In Phase I metabolism, the biotransformation of d-limonene and Terpinolene monoconstituent are catalyzed by NADPH-dependent cytochrome P450 (CYP). d-Limonene (monocyclic hydrocarbon) has been shown to be substrate (upon repeated administration) and competitive inhibitor of the isoenzyme, specifically CYP2B1 and CYP2C11 (Miyazawa et al., 2002). Limonene has also been shown to induce the members of the CYP2B family in several studies (Maltzman et al., 1991; Hiroi et al., 1995).

Excretion:

Having a molecular weight lower than 300, TERPINOLENE MONOCONSTITUENT is expected to be mainly excreted in urine and no more than 5-10% may be excreted in bile. Urinary excretion is supported by metabolism data described above but also by the effects identified in the repeated dose toxicity study with reproduction/developmental toxicity screening test. At 3000 and 9000 ppm, partly reversible changes in kidney (tubular degeneration/regeneration, hyaline droplets and granular casts) were observed in main phase and recovery males. Although these effects are specific to male rat, they are suggestive of excretion via urine of the parent molecule or its metabolites.

Clearance from the blood was 1.1 litre/kg body weight per hour in males exposed for 2 hours to d-limonene at 450 mg/m3 (Falk Filipsson et al., 1993). About 60% of the radiolabelled d-limonene administered by inhalation was recovered from the urine, with 5% from feces and 2% from expired CO₂in rats (Igimi et al., 1974). Following the inhalation exposure of volunteers to d-limonene at 450 mg/m3 for 2 hours, three phases of elimination were observed in the blood, with half-lives of about 3, 33, and 750 minutes, respectively (Falk Filipsson et al., 1993). About 1% of the amount taken up was eliminated unchanged in exhaled air, whereas about 0.003% was eliminated unchanged in the urine. When male volunteers were administered (per os) 1.6 g [14C]d-limonene, 50–80% of the radioactivity was eliminated in the urine within 2 days with less than 10% appearing in the feces (Kodama et al., 1976). Limonene has been detected, but not quantified, in breast milk of non-occupationally exposed mothers (Pellizzari et al., 1982).

 

Accumulative potential:

Terpinolene monoconstituent has a low water solubility (< 100 mg/L) and high log Kow (>4) therefore it has affinity to adipose tissues; however, bioaccumulation is not expected to occur, since it is efficiently metabolized to yield oxygenated metabolites that are subsequently conjugated with glucuronic acid and excreted mainly in the urine.

 

Information on 1,8-Cineol:

Only information on 1,8-Cineol metabolism are available. In an in vivo metabolism study in rats exposed by oral gavage, 1,8-Cineol was metabolised to hydroxylated derivatives such as 2-hydroxy cineole and 3-hydroxy cineole which were excreted as conjugates. It is rather difficult to predict the sequence of reactions taking place during the biotransformation of cineole. However, one can envisage the formation of 1,8-dihydroxy-10-carboxy-p-methane through the intermediary of p-methane 1,8-diol and further metabolism is possibly initiated by the oxygenation of the C-10 methyl group resulting in the formation of p-methane-1,8,10-triol which undergoes stepwise oxidation to the corresponding aldehydes and then to an acid. The opening of the ether bridge in cineole could result in the formation of a p-menthanoid cation with a positive charge either at C-1 or C-8 which further gets readily neutralized by the attack of a hydroxide ion to yield p- methane-1,8-diol.

 

Also, in an in vitro study with human liver microsomes, 1,8-Cineol was oxidized to the only metabolite 2-exo-hydroxy-1,8-cineole.

 

Information on TERPINOLENE MULTICONSTITUENT:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422, main phase males exposed to TERPINOLENE MULTICONSTITUENT showed a slight increase in absolute and body weight relative liver weights, compared to controls. Centrilobular hepatocellular hypertrophy was observed in males with an incidence and/or severity proportional to the dose administered at all dietary inclusion levels. Liver of females receiving 7500 ppm also showed minimal centrilobular hypertrophy. After fourteen days of recovery, liver morphology was considered to have returned to normal. The hepatocellular hypertrophy observed was considered as an adaptive metabolic response of the liver to the presence of a xenobiotic. In kidneys, treatment-related lesions characterized by tubular degeneration and regeneration, granular casts, interstitial fibrosis and mixed cell infiltration, mainly of the proximal portion of the nephrons, were observed in males receiving 7500 ppm. The lesions were suggestive of alpha 2 μ-globulin nephropathy.

These observations are suggestive of metabolism of TERPINOLENE MULTICONSTITUENT in the liver and excretion in urine; they confirm the data available on Terpinolene monoconstituent for which similar effects were observed at lower doses on liver and kidneys in the same type of study.

 

References:

Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.M., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2008. A toxicologic and dermatologic assessment of cyclic acetates when used as fragrance ingredients. Food Chem. Toxicol. 46 Suppl 12, S1–27.

Ishida, T., Toyota, M., Asakawa, Y., 1989. Terpenoid biotransformation in mammals. V. Metabolism of (+)-citronellal, (+-)-7-hydroxycitronellal, citral, (-)-perillaldehyde, (-)-myrtenal, cuminaldehyde, thujone, and (+-)-carvone in rabbits. Xenobiotica 19, 843–855.

 

Alicyclic Primary Alcohols, Aldehydes, Acids, and Related Esters,WHO Food Additives Series 50

 

Falk Filipsson, A., 1998. Concise International Chemical Assessment Document 5 - Limonene, 32 Chapitre 7. Comparative kinetics and metabolism in laboratory animals and humans.

Flavor and Fragrance High Production Volume Chemical Consortia, 2006, HPV Monoterpene Hydrocarbons

 

Madyastha KM, Chadha A. 1986 Metabolism of 1,8-cineole in rat: its effects on liver and lung microsomal cytochrome P-450 systems. Bull Environ Contam Toxicol. Nov;37(5):759-66.

 

Miyazawa M, Shindo M. 2001 Biotransformation of 1,8-cineole by human liver microsomes. Nat Prod Lett.;15(1):49-53.