Reaction mass of 1-methyl-4-(1-methylethylidene)cyclohexyl acetate and p-menth-1-en-8-yl acetate

Endpoint summary

Description of key information

The constituents of Terpinyl Acetate multi will not be as such in the sytemic circulation because the half life in aqueous solutions is <= 10 minutes (at pH 2,4 and 7) and in plasma it is < 60 minutes.

It is anticipated that Terpineol multi constituents will be the key metabolites based on information from Linalyl acetate.

The oral, dermal and inhalation absorption is considered to be 100% for all routes.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

Terpinyl Acetate multi - experimental information on metabolism/degradation

The stability of Terpinyl Acetate multi was evaluated in rat plasma and blood in two separate experiments. Both of these experiments were performed in duplicate. In both experiments rat k2 -EDTA matrix was spiked with analyte solution to achieve an initial concentration at 50 ug.ml. The spiked matrix was kept at 37oC. The spiked matrix samples were aliquoted at different time points: 0, 1,3, 6, 9, 15, 30, 60, 120 and 240 minutes. In addition, the stability of Terpinyl Acetate multi was evaluated in aqueous solutions of different pH: 2,4 and 7.

Results: Two peaks were seen in the chromatogram: Terpinyl Acetate alpha and gamma presumably being the two key constituents. The substance decreased steadily. DT50 in plasma was calculated to be ca 50 minutes for both constituents. The DT50 regression line for plasma is modelled from the equation: y=A1 (response value)*(exp(-x/t1)+y: A=309706 (response value)*exp(-x/t1=73.1)+y=0 = 38592, k=0.0137 and r2 0.866. In the control where proteins including esterases were denaturated no decrease of Terpinyl Acetate alpha or gamma was seen. The degradation product(s) could not yet be captured in the chromatograms and was considered to be due to the limitation of the method. Further work is ongoing to detect the degradation products. In aqueous solutions the decrease of Terpinyl Acetate multi was <=10 minutes. Conclusion: Terpinyl Acetate is not stable in plasma and aqueous solutions and therefore no Terpinyl Acetate multi will be in the systemic circulation. Due to the absence of any metabolite/degradation product further work is needed to know what these are. Further work is currently initiated.

Terpinyl Acetate multiand its toxico-kinetic behaviour

Introduction

The constituents of Terpinyl Acetate multi are tertiary acetic-esters attached to an unsaturated cyclohexyl ring, with a methyl group attached to the unsaturated bond at the para-position. It has a melting point of -20^oC and a molecular weight of 196 g/mol, which do not preclude absorption. The substance has a low volatility of 3.5 Pa. Terpinyl Acetate multi is natural occurring in a wide variety of foods, fruits, spices and tea.

The toxico-kinetic information presented is mainly based on Terpinyl Acetate alpha (being its main constituent) but includes also its other constituents. In the table below the structural isomers are presented (Table 1). In this table a complete overview is presented for the acetates and the alcohols because read across is applied between acetates and between acetates and alcohols for a number of endpoints.

Structural similarities and differences in the Terpinyl Acetate constituents: the constituents of Terpinyl Acetate multi are structural isomers. The functional acetate group is present in all constituents. The difference is that Terpinyl Acetate alpha has its acetate on the opposite side compared to Terpinyl Acetate gamma and beta (Table 1).

Table 1 The constituents of alpha-Terpineol, Terpineol multi, alpha-Terpinyl Acetate, Terpineol Acetate multi

Terp-inoids in %	Alpha-Terpineol	Gamma-Terpineol	cis-beta-Terpineol	trans-beta-Terpineol	Alpha-Terpinyl Acetate	Gamma-Terpinyl Acetate	cis-beta- Terpinyl Acetate	trans-beta Terpinyl Acetate Tab
Cas nu	98-55-5	586-81-2	138-87-4 Generic	138-87-4, Generic	80-26-2	10235-63-9	20777-47-3	59632-85-8
Alpha-Terpineol	85	11	< 1	< 1	0	0	0	0
Terpineol multi	60	29	9	3	0	0	0	0
Alpha-Terpinyl Acetate	0	0	0	0	87	9	1	1
Terpinyl Acetate multi	0	0	0	0	64	20	7	4

Kinetics

Absorption, Oral: The relatively low molecular weight, the moderate octanol/water partition coefficient (Log Kow 4.4) and water solubility (36 mg/l) would favour absorption through the gut. According to Martinez and Amidon (2002) the optimal log Kow for oral absorption falls within a range of 2-7. This indicates that the oral absorption is high.

Skin: Dermal absorption is likely to occur, based on its molecular weight (196 g/mol) and its physico-chemical characteristics: log Kow (4.4) and water solubility (36 mg/l). The optimal MW and log Kow for dermal absorption is < 100 g/mol and in the range of 1-4, respectively (ECHA guidance, 7.12, Table R.7.12-3). Terpinyl Acetate alpha is just outside optimal range and therefore the skin absorption is not expected to exceed the oral absorption.

Lungs: Absorption via the lungs is also indicated based on these physico-chemical properties. The octanol/water partition coefficient (4.4) 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.

ForTerpinyl Acetatethe B/A partition coefficient would result in:

Log P (BA) = 6.96 – 1.04 x 0.56 – 0.533 x 4.4 – 0.00495 x 196= 3.06

This means that the constituents ofTerpinyl Acetatemulti have a 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. Despitethe substancebeing somewhat out of the applicability domain and the exact BA may not be fully correct, it can be expected that the substance will be readily absorbed via the inhalation route.

Distribution of the parent substance: The moderate water solubility of the 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 extensive metabolism, the substance as such does not accumulate in the body fat.

Metabolism Phase 1, De-esterification of the acetate: The constituents of Terpinyl Acetate multi are expected to be de-esterified into the constituents of Terpineol multi and simultaneously be metabolised as Terpineol multi. Experimentally is shown that Terpinyl Acetate multi decreases by half within ca. 10 minutes under acidic and neutral aqueous conditions. The substance decreased with 50% within an hour when incubated in rat blood (IFF, 2020). The substance was recovered in denaturated plasma indicating that carboxyl esterases played a role in the degradation in plasma/blood. This means that Terpinyl Acetate multi will not exist as such in the systemic circulation after absorption, because of acidic aqueous conditions in the stomach and intestinal tract and esterases present in liver and blood.

Within these experiments, carried out both in plasma and acidic condition, there were no degradation products found in the GC-MS MS chromatograms and further work is needed to identify these to support this reasoning.

Now only the degradation of Terpinyl Acetate multi is experimentally shown, the metabolic pathways of Terpinyl Acetate multi as presented in literature is needed to complete the kinetics of Terpinyl Acetate multi. In the EFSA review on Tertiary saturated and unsaturated alcohols and esters (2011) they present the Terpinyl Acetate metabolism into Terpineol. They support this pathway with the experimental metabolic pathways of Linalyl Acetate considered to be an analogue for Terpinyl Acetate, because they only differ in the alkene chain being open or closed, respectively. Based on this work and from Wu et al. (2010) Linalyl acetate can be zipped into the Terpinyl Acetate pathway (Fig. 1). Linalyl Acetate is a close analogue of Terpinyl Acetate multi. The structural difference is that Linalyl Acetate has a straight alkyl chain, while Terpinyl Acetate has these carbons organised in a ring. During metabolism the terpinyl ring can be formed from Linalyl by closing the alkene chain (Fig. 1) supporting a common metabolite. The EFSA summary on Linalyl acetate kinetics is presented below.

EFSA, 2011: “Two hydrolysis studies on a supporting substance, linalyl acetate, were found.

In an in vitrohydrolysis study, linalyl acetate was easily hydrolysed in water and simulated gastric and pancreatic fluids. The mean half-lives for linalyl acetate hydrolysis were 5.5 and 52.5 min in gastric and pancreatic fluids, respectively (Hall, 1979). In neutral gastric juice, linalyl acetate is slowly (t½ = 121 min) hydrolysed to a mixture of linalool and the ring-closed isomer alpha-terpineol.

In acidic artificial gastric juice, linalyl acetate is rapidly hydrolysed (t½ < 5 min) to yield linalool (Buck & Renwick, 1998). Linalyl acetate was slowly hydrolysed (t½ = 153 – 198 min) in intestinal fluid with or without pancreatin. Linalyl acetate also hydrolyses in homogenates of rat intestinal mucosa, blood and liver, but at rates much slower than in acidic gastric juice (rate constant for hydrolysis, k = 0.01 to 0.0055 min-1 vs. > 5 min-1 in gastric juice). Based on these observations it can be concluded that Linalyl acetate hydrolyses in gastric juice to yield linalool and acetic acid (Buck & Renwick, 1998). Further, it has been demonstrated that linalyl acetate can be hydrolysed to linalool in in vitrostudies after incubation with rat caecal flora (Rahman, 1974a)”.

Table 2 Half-life (DT50 or T ½) are shown for Terpinyl Acetate multi and Linalyl acetate based on experimental information

	Terpinyl Acetate multi T1/2 minutes (k elimination in brackets)	Linalyl acetate T1/2 minutes (k elimination in brackets)
Cas no.	8000-41-7 (generic)	115-95-7
Structural features of metabolism
MW	196	196
Acidic artificial gastric juice	8.8 (water, pH2)	5.5
Pancreatic fluids	7.5 (water, pH4)	52.5
Neutral gastric fluids	10.3 (water, pH7)	121
Intestinal mucosa, blood and liver	55 (plasma, k=0.0137)	T1/2 not reported (k=0.01 to 0.0055)
Reference	IFF, 2020 (CRL)	Hall, 1979 Unpublished report; As sited in EFSA, 2011.

From Table 2 the half-lives of Terpinyl Acetate and Linalyl acetate can be compared in various media. At pH 2 the half-lives are very similar. At pH 4 and 7 the aqueous solutions with Terpinyl Acetate have a shorter half-life compared to Linalyl acetate. The aqueous acidic conditions for Terpinyl Acetate alpha show somewhat faster degradation at higher pHs compared to mammalian gastric juices because the bioavailability may be somewhat less in gastric juices. The kinetic coefficient (k elimination) in plasma and intestinal mucosa/blood and liver for both substances are roughly in line.

With this information the degradation products can be characterised by using Linalyl acetate as a surrogate, for which information on metabolism and degradation products are available and can be integrated with the Terpinyl acetate as shown below. The presence of the type of metabolite is qualitative.

Metabolism Phase 1- Other metabolic steps than acetate cleavage: As presented in Fig. 1 hydroxylation of the ring and/or oxidation of the head allyl-methyl group and/or the reduction of the tertiary alcohol can occur. The oxidation of the head allyl-methyl group can result in acidic terminal groups. An overview of terpinoid metabolism by P450 enzymes is shown by Banerjee and Hamberger (2017) showing a vast number of possibilities. The ones relevant for specifically the constituents of Terpinyl Acetate multi are presented in Fig. 1, which are compiled from Wu et al. (2010), RIFM (2016), Madyastha and Srivastan (1988) and NCI,DCPC (1996). Madyastha and Srivastan have shown in vivo the formation of Oleuropeic acid and by reduction of the tertiary alcohol Perillic acid can be formed (Fig. 1). However in the study of RIFM (2016) such acidic metabolites were not found/determined. NCI, DCPC (1996) showed that Limonene can result in Perillic acid.

 

Fig. 1     A simplistic scheme of the metabolic pathway of TerpinylAcetate alpha and gamma is presented. The Terpinyl Acetate gamma is presented to show its structural similarity with Linalyl Acetate (closed versus open ring, respectively). The substance after acetate cleavage becomes a tertiary alcohol (Cas no 98-55-5) and Acetic acid. This tertiary alcohol can be reduced into a double bond. It is also possible that the methyl group at the para-position can be hydroxylated and/or further oxidised into an acid (See text for references and further explanation). The + in the structure of the intermediate between Linalool and Limonene indicates the carbocation.

Phase 2 metabolisation: The Terpinyl alcohols will be glucuronidated and subsequently excreted (WHO, 2000, RIFM, 2016). Acetic acid is an endogenous component of body; it will be consumed in the Krebs cycle.

Metabolic overload due to high dosing likely occurring for Terpineol:In the Terpineol multi (MW 154) ECHA dossier the kinetics of Terpineol are presented in detail, summarizing available data and thoughts:https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/22822/7/2/1. From this information in which SD-rats were exposed to 100, 250, 600 or 750 mg/kg bw Terpineol Alpha, the AUC of Terpineol glucuronide increased almost dose-proportionally from 100 to 600 mg/kg, whilst at 750 mg/kg a more than dose-proportional increase was observed, being indicative for overloading the metabolic pathways.

Distribution of the metabolite:The hydroxylated, glucuronidated or acidic metabolites are water soluble and will be distributed in the blood and will not accumulate in the body fat.

Excretion:The primary route of excretion is expected to be through the urine because the substance will be extensively metabolised into more hydrophilic groups.

Discussion

The substance is expected to be readily absorbed, orally and via inhalation, based on the human toxicological information and physico-chemical parameters and further information from related substances as discussed in the WHO (2000) review on this material. The substance also is expected to be absorbed dermally based on the physico-chemical properties. The MW and the log Kow are higher than the favourable range for dermal absorption but significant absorption is likely. 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.

Conclusion

Terpinyl Acetatemulti’s constituents are expected to be readily absorbed via the oral and inhalation route and somewhat lower via the dermal route based on toxicity information and physico-chemical data. In the present case 100% oral and 100% dermal absorption will be used not to underestimate the dermal route. Also for inhalation 100% absorption will be used. Complete de-esterification is expected to occur on which bases Terpinyl Acetate multi will have similar systemic effects as Terpineol multi.

References

Banerjee, A., and Hamberger, B., 2017,P450s controlling metabolic bifurcations in plant terpene specialized metabolism, Phytochemistry reviews, 17, 81-111,https://link.springer.com/content/pdf/10.1007%2Fs11101-017-9530-4.pdf

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

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

EFSA, 2011, Aliphatic, alicyclic and aromatic and unsaturated tertiary alcohols, aromatic tertiary alcohols and their esters from chemical group 6 and 8;https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2011.1847

Madyastha, K.M., Shrivastan, V., 1988, Biotransformation of alpha-Terpineol in the rat: Its effects on the liver microsomal cytochrome P-450 system, Bull. Environ. Contam. Toxicol., 41, 17-25.

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.

NCI and DCPC, Chemoprevention Brancy and Agent Development Committee, 1996, Clinical Developmental plan: l-Perillic alcohol, Journal of Cellular Biochemistry, 26S: 137-148.

RIFM, 2016, Alpha Terpineol: Metabolism in rats after single oral (gavage) exposure, unpublished report, RIFM no 70825.

WHO, 1999, Food additive series 42, 1999, Evaluation of certain food additives, Aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related compounds,http://www.inchem.org/documents/jecfa/jecmono/v042je17.htm

WHO, 2000, Evaluation of certain food additives, Technical Report Series 891, page 51-54,http://whqlibdoc.who.int/trs/WHO_TRS_891.pdf

Wu, S., Blackburn, K., Amburgey, J., Jaworska, J., Federle, F., 2010. A Framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments. Regul. Toxicol. Pharm. 56, 67–81.