2,6-dimethyloct-7-en-2-yl acetate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March-June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

other: hydrolysis and degradation of geranylacetate extra in plasma, liver and gastrointestinal tract

Qualifier:

no guideline available

Principles of method if other than guideline:

The hydrolysis and degradation of Geranylacetat Extra in plasma, liver and gastrointestinal tract was investigated. To determine hydrolysis in either compartment, the test substance was incubated with plasma and liver-S9 fraction from rat as well as in gastric-juice simulant and intestinal-fluid simulant. After incubation, the proteins were precipitated and the amount of remaining substrate was analysed in the supernatant by GC/FID.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

other: not applicable; in vitro test

Statistics:

For calculation of the hydrolysis (turn over), peak areas of the substrates of active incubation (AI) and heat deactivated control (HDC) were used. Buffer controls were used to calculate the substrate recovery of the HDC and t=0 control.

Calculation of the turn over in plasma, liver- S9 fraction and intestine-fluid simulant:
% turn-over = 100 * (peak area (HDC) – peak area (AI)) / peak area (HDC)

Calculation of the turn-over in gastric-juice simulant:
% turn-over = 100 * (peak area (t=0) – peak area (AI)) / peak area (t=0)

Geranylacetate Extra hydrolyzed within 0.5h almost completely in rat plasma, liver S9 fraction of rats and intestinal- fluid simulant under the test conditions used. Hydrolysis was demonstrated by the decrease of Geranylacetate Extra as well as by the formation of its hydrolysis product Geraniol Extra. Although the recovery of Geranylacetate Extra was tentatively low in respective controls, the quantitative amount of Geraniol Extra formed was correlated to the nominal substrate concentration of Geranylacetate in incubates and proved a high metabolic turn over. It is assumed that the low recoveries of Geranylacetate in HDC controls are based on potential evaporation of the test substance. In t=0 controls, low recoveries are due to partial hydrolysis, since Geraniol Extra is generally already detectable

in these controls, proving that hydrolysis was faster than the stopping process after addition of the test substance to the system. In gastric fluid simulant, the degradation of Geranylacetate Extra was slower and was calculated to be about 50 % after an incubation period of 2h. The degradation of Geranylacetate Extra yielded maximum degradation rates of ≥211.8 [μmol/L*h], ≥2.7 [μM/g liver equivalent*h], ≥21.3 [μM/g pancreas lipase (15-35 U/mg)*h] and 93.2 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively. These maximum degradation rates of Geranylacetate Extra were in the same order of magnitude than the maximum degradation rates determined for the applied positive control Benzyl benzoate and demonstrate a fast hydrolysis of Geranylacetate Extra in gastrointestinal tract, liver and plasma.

Conclusions:

The current in vitro data demonstrate that Geranylacetate Extra is hydrolyzed within 0.5 h to Geraniol almost completely in rat plasma, liver S9 fraction of rats and intestinal- fluid simulant under the test conditions used. In gastric fluid simulant the degradation of Geranylacetate Extra was slower and was calculated to be about 50 % after an incubation period of 2 h. The degradation of Geranylacetate Extra yielded maximum degradation rates of ≥211.8 [μmol/L*h], ≥2.7 [μM/g liver equivalent*h], ≥21.3 [μM/g pancreas lipase (15-35 U/mg)*h] and 93.2 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively.
These maximum degradation rates of Geranylacetate Extra were in the same order of magnitude as the maximum degradation rates determined for the positive control Benzyl benzoate.

Executive summary:

To determine hydrolysis in either compartment, the test substance was incubated in duplicates at a nominal concentration of 250 μM for 0.5, 1 and 2h at 37°C in plasma, liver S9 fraction of rats, gastric-juice simulant and intestinal-fluid simulant including pancreas lipase. After incubation, proteins in incubates were precipitated by the addition of one volume acetone and the amount of remaining

substrate was analysed in the supernatant by GC/FID. Heat deactivated controls (HDC, with the exception of gastric juice simulant) and controls, directly stopped after addition of test substance (t=0 control) served as control samples. A buffer control or medium (BC, test substance in the incubation buffer; MC, test substance in the incubation medium) was used for calculation of recoveries in the HDC and t=0 controls. Additionally recovery of controls was related to the target concentration of the test substance in the in vitro incubate (250 μM).

Positive controls were performed with Benzyl benzoate at a concentration of 250μM. In rat plasma and liver S9 fraction of rats, Benzyl benzoate was hydrolyzed after 0.5 h almost completely, resulting in a metabolic turnover of 97 and 99%, respectively. In the in vitro systems of gastrointestinal tract, hydrolysis was slower, yielding maximum metabolic turn over values of 43 and 51 % after 2h for intestine fluid simulant and gastric fluid simulant, respectively. The maximum degradation rates of Benzyl benzoate were ≥182.3 [μmol/L*h], ≥4.9 [μM/g liver equivalent*h], 27.6 [μM/g pancreas lipase (15-35 U/mg)*h] and 84.6 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively. Based on these results, the validity of the applied in vitro systems as well as of the chosen incubation conditions was clearly demonstrated.

The current in vitro data demonstrate that Geranylacetate Extra is hydrolyzed within 0.5 h to Geraniol almost completely in rat plasma, liver S9 fraction of rats and intestinal- fluid simulant under the test conditions used. In gastric fluid simulant the degradation of Geranylacetate Extra was slower and was calculated to be about 50 % after an incubation period of 2 h. The degradation of Geranylacetate Extra yielded maximum degradation rates of ≥211.8 [μmol/L*h], ≥2.7 [μM/g liver equivalent*h], ≥21.3 [μM/g pancreas lipase (15-35 U/mg)*h] and 93.2 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively.

These maximum degradation rates of Geranylacetate Extra were in the same order of magnitude as the maximum degradation rates determined for the positive control Benzyl benzoate.

Description of key information

For Dihydromyrcenyl acetate no experimental toxico-kinetic data are available for assessing adsorption, distribution, metabolism and excretion of the substance. For Geranyl acetate metabolism information is available which will be used to support the read across to Dihydromyrcenyl acetate from Dihydromyrcenol. The executive summpary for the Geranyl acetate is presented in the additional information section.

The kinectic informattion for Dihydromyrcenyl acetated is based on effects seen in the human health toxicity studies and physico-chemical parameters. The substance is expected to be readily absorbed via the oral and inhalation route and somewhat lower via the dermal route. Using the precautionary principle for route to route extrapolation the final absorption percentages derived are: 50% oral absorption, 50% dermal absorption and 100% inhalation absorption.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

Before the overall toxico-kinetic information on Dihydromyrcenyl acetate will be presented the in vitro metabolism study of Geranyl acetate will be summarised.

Geranyl acetate metabolism in vitro

To determine hydrolysis in either compartment, the test substance was incubated in duplicates at a nominal concentration of 250 μM for 0.5, 1 and 2h at 37°C in plasma, liver S9 fraction of rats, gastric-juice simulant and intestinal-fluid simulant including pancreas lipase. After incubation, proteins in incubates were precipitated by the addition of one volume acetone and the amount of remaining

substrate was analysed in the supernatant by GC/FID. Heat deactivated controls (HDC, with the exception of gastric juice simulant) and controls, directly stopped after addition of test substance (t=0 control) served as control samples. A buffer control or medium (BC, test substance in the incubation buffer; MC, test substance in the incubation medium) was used for calculation of recoveries in the HDC and t=0 controls. Additionally recovery of controls was related to the target concentration of the test substance in the in vitro incubate (250 μM).

Positive controls were performed with Benzyl benzoate at a concentration of 250μM. In rat plasma and liver S9 fraction of rats, Benzyl benzoate was hydrolyzed after 0.5 h almost completely, resulting in a metabolic turnover of 97 and 99%, respectively. In the in vitro systems of gastrointestinal tract, hydrolysis was slower, yielding maximum metabolic turn over values of 43 and 51 % after 2h for intestine fluid simulant and gastric fluid simulant, respectively. The maximum degradation rates of Benzyl benzoate were ≥182.3 [μmol/L*h], ≥4.9 [μM/g liver equivalent*h], 27.6 [μM/g pancreas lipase (15-35 U/mg)*h] and 84.6 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively. Based on these results, the validity of the applied in vitro systems as well as of the chosen incubation conditions was clearly demonstrated.

The current in vitro data demonstrate that Geranylacetate Extra is hydrolyzed within 0.5 h to Geraniol almost completely in rat plasma, liver S9 fraction of rats and intestinal- fluid simulant under the test conditions used. In gastric fluid simulant the degradation of Geranylacetate Extra was slower and was calculated to be about 50 % after an incubation period of 2 h. The degradation of Geranylacetate Extra yielded maximum degradation rates of ≥211.8 [μmol/L*h], ≥2.7 [μM/g liver equivalent*h], ≥21.3 [μM/g pancreas lipase (15-35 U/mg)*h] and 93.2 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively. These maximum degradation rates of Geranylacetate Extra were in the same order of magnitude as the maximum degradation rates determined for the positive control Benzyl benzoate.

 

The toxico-kinetic behaviour of Dihydromyrcenyl acetate (CAS nr. 53767-93-4)

Introduction

The test material Dihydromyrcenyl acetate (Cas no 53767-93-4) has a branched alkene chain to which an acetate ester is attached. It is a liquid with a molecular weight of 198.3, water solubility (WS) of 6.1 mg/L and a log Kow of 4.9 that does not preclude absorption. The acetate can be hydrolysed. The substance has a low volatility of 14.4 Pa.

Absorption

Oral:The relatively low molecular weight and the moderate octanol/water partition coefficient (Log Kow 4.9) 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 shows that Dihydromyrcenyl acetateis 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 (198.3), log Kow (4.9) and water solubility (6.1 mg/L), indicate that (some) dermal absorption is likely to occur. The optimal MW and log Kow for dermal absorption is < 100 and in the range of -1 to +4, respectively (ECHA guidance, 7.12, Table R.7.12-3) and therefore the dermal absorption will be somewhat (s)lower compared to oral absorption. Therefore the dermal absorption will not exceed the oral absorption and considered to be =<50%.

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 (14.4 Pa), the octanol/water partition coefficient (4.9), indicates that inhalation absorption is possible.

 

Log PBA = 6.96 – 1.04 Log (VP) – 0.533 (Log) Kow – 0.00495 MW, for the substance this results in:

 

Log P (BA) = 6.96 – 1.04 Log (14.4)– 0.533*4.9 – 0.00495*198 = 2.2

 

This means that the substance has 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. Despite the substance 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

The moderate water solubility of the test substance indicates some distribution in the body via the water channels. The log Kow suggests that the substance would pass through the biological cell membrane. The substance does not have a bioaccumulating potential based on a BCF of 156 derived from the analogue Verdox, also an acetic ester, and based on metabolisation of the acetate.

Metabolism

There are no experimental data on the metabolism of Dihydromyrcenyl acetate. The substance metabolises into Dihydromyrcenol and acetic acid as was experimentally established for Geranyl acetate (see IUCLID section 7.1.1). The double bond at the end is prone to oxidation and an epoxide may be an intermediate metabolite. After ester cleavage Dihydromyrcenol will be conjugated. One pathway will be the conjugation at the tertiary alcohol with alpha-2u globulin because this protein sediments in the kidneys after transport of Dihydromyrcenol as was seen in the repeated dose toxicity studies.

Fig. 1 The first metabolic step of Dihydromyrcenyl acetate into Dihydromyrcenol and acetic acid.

 

Excretion

Because of the moderate water solubility and the relatively low molecular weight, Dihydromyrcenyl acetate and its metabolites are expected to be excreted mainly via urine, and possibly also via the bile. Any unabsorbed substance will be excreted via the faeces.

Discussion

Dihydromyrcenyl acetate is expected to be readily absorbed, orally and via inhalation, based on the human toxicological information and physico-chemical parameters. The substance also is expected to be absorbed dermally based on the physic-chemical properties. The MW and the log Kow are higher than the favourable range for dermal absorption but significant absorption is likely.

In view of the absence of adverse effect in the repeated dose / reproscreen study route to route extrapolation is not needed and therefore conversion to the inhalation and dermal route is not needed.

Conclusion

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

References

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

 

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.