Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: FDA/AAPS guidelines

Version / remarks:

See "Principles of method if other than guideline"

Principles of method if other than guideline:

Testing was performed according to Skelly et al., "FDA and AAPS report of the workshop on principles and practices of in vitro percutaneous penetration studies: relevance to bioavailability and bioequivalence", Pharmaceutical Research 1987, 4, 265-267.
In short, full thickness human skin samples, resulting from the breast or the abdomen of cosmetic surgery patients, were heat separated and the epidermal membranes were used in the study. The epidermal membranes were mounted on filter paper supports. The supports, with the membranes, were then placed onto diffusion cells and trimmed to size. The integrities of the membranes were determined by using tritiated water. Twenty mL of a 1% test item solution in ethanol was applied to the surface of the membranes. At 2, 8, 24, 36, and 48 hours 200 mL samples of the receptor fluid (50% ethanol/ water solution) were taken from the receptor chamber and analysed by liquid scintillation chromatography. Evaporative loss of the test item was also determined. In parallel, PTFE sheet mounted in diffusion cells (at 32°C) was closed with 20 µL of 1% solution of the radiolabelled test item in ethanol. Six cells were prepared. Following dosing, one cell was dismantled at each of the following timepoints: 1, 2, 4, 8, 24 and 48 hours. The PFTE sheet was removed and washed twice with methanol (first with 10 mL, then 5 mL). The donor chamber was washed with 10 mL methanol. A sample of each wash solution was submitted to analysis by LSC that allowed the total remaining radiolabel at each time point to be determined.

GLP compliance:

not specified

Specific details on test material used for the study:

Vapour pressure (mm Hg, 20°C) of the test item: 0.002 Pa

Radiolabelling:

yes

Remarks:

0.2mCi methyl artrarate, [1-14C], specific activity 46.02 mCi/mmol, radiochemical purity 98.01%

Doses:

1%

Key result

Time point:

24 h

Dose:

1%

Parameter:

percentage

Absorption:

13.8 %

Key result

Time point:

48 h

Dose:

1%

Parameter:

percentage

Absorption:

20.3 %

Conversion factor human vs. animal skin:

Not relevant, human skin samples were used.

Recovery of the test item at 48 hours was 94.8%:

Surface wipe: 64.3%; tape strips: 4.6%; remaining epidermis: 3.8%; receptor phase: 20.3%; donor chamber: 1.8%.

There was no measured loss for the test item due to evaporation.

The liquid fragrances tested permeated the skin fairly rapidly, but the permeation subsequently plateaud as the donor phase became depleted through permeation. The permeation rate of methyl atrarate was slower and some plateauing occurred.

Conclusions:

Percutaneous absorption of the test item was assessed by an in vitro skin penetration study. At 24 and 48 hours, 13.8% and 20.3% of the test item (radiolabelled, applied in 1% solution in ethanol) permeated through the membrane. Total recovery at 48 hours was 94.7%, there was no loss due to evaporation.

Executive summary:

Percutaneous absorption of the test item was assessed by an in vitro skin penetration study. At 24 and 48 hours, 13.8% and 20.3% of the test item (radiolabelled, applied in 1% solution in ethanol) permeated through the membrane. Total recovery at 48 hours was 94.7%, there was no loss due to evaporation.

Description of key information

A dermal absorption study is available in which dermal absorption was 20%. Veramoss is expected to be readily absorbed via the oral and inhalation route and somewhat less via the dermal route based on physico-chemical information. The 20% dermal absorption information is not critical and therefore the resulting absorption percentages 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

Dermal absorption study (uses as supporting information)

Percutaneous absorption of the test item was assessed by an in vitro skin penetration study according to guidelines. At 24 and 48 hours, 13.8% and 20.3% of the test item (radiolabelled, applied in 1% solution in ethanol) permeated through the membrane. Total recovery at 48 hours was 94.7%, there was no loss due to evaporation.

The toxico-kinetic behaviour of Veramoss (CAS no. 4707-47-5)

Introduction

The test material Veramoss (Cas no 4707-47-5) is an ester. It is a solid with a molecular weight of 196,water solubility (WS) of 18 mg/L and a log Kow of 2.6, indicative for good absorption. The substance has a low vapour pressure of 0.002 Pa. The substance is expected to hydrolyse in water fairly quickly because the ester is adjacent to an electrophilic aromatic ring to which OH groups are attached, which increases the speed of hydrolysis.

Absorption

Oral: In absence of effects in the acute oral toxicity study no conclusion can be drawn on the absorption potential. Also in the repeated dose-reproscreen study only minimal effects were seen. Therefore the oral absorption is based on the physico-chemical properties. The relatively low molecular weight and the moderate octanol/water partition coefficient (Log Kow 2.6) favour absorption through the gut. This means that Veramossis likely to be absorbed orally and therefore the oral absorption is expected to be much higher > 50%.

Skin: A skin absorption study is available showing 20% dermal absorption. From the acute dermal toxicity study the skin absorption cannot be estimated in absence of any effects at >= 2000 mg/kg bw. Based on the physico-chemical characteristics of the substance, being a solid, its molecular weight (196), log Kow (2.6) and water solubility (18 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). In view of the physico-chemical properties and the skin absorption found the dermal absorption it can be seen that the dermal absorption will not exceed the oral absorption and a value of 50% can be used conservatively.

Lungs: Absorption via the lungs is also indicated based on these physico-chemical properties. The exposure via the inhalation route may occur where the substance is used in its pure granular form because 70% is < 100 um and its respirable fraction (< 10 um) is 5.3%. When used in a mixture the substance the inhalation route is thought negligible, because of its low volatility (0.002 Pa, being < 0.01Pa). The octanol/water partition coefficient (2.6) 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. The regression line is only valid for substances with a vapour pressure >100 Pa. In view of the large discrepancy between 100 (model) and 0.002 Pa (substance) it was thought not reliable to calculate this blood/air partition coefficient.

Distribution

The moderate water solubility of the test substance is indicative for 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 log Kow and the expected metabolism the substance as such is not expected to accumulate in the body fat. The substance is expected to be distributed as its acid in view of the expected hydrolysis and metabolism at all routes of entry.

Metabolism

There are no actual data on the metabolism of Veramoss but it can be predicted that Veramoss will metabolise in the gut and/or in the liver into its respective Veramoss-acid and methanol in view of it’s the electrophilic activity adjacent to the ester and the presence of carboxyl esterases abundantly available (Toxicological handbooks and e.g. Yamada et al., 2013). The Veramoss- acid will be dissociated in the blood because the acid has a pKa of 3.21 and fast distribution as well as excretion is expected. Quinone type metabolites (the aromatic ring containing two double bonds) seem rather unlikely with the ester bond on the other end. According to the OECD Toolbox such quinone type can only occur when there is an OH group or a CH3 group at either end, which is not the case.

In addition to this ester metabolisation the P450 system can hydrolyse the methyl groups attached to the ring and/or aromatic ring (see pointing arrows, in Fig. 1)

Figure 1 Prediction of the metabolic pathway of Veramoss (OECD Toolbox and Toxtree v2.6.6: the ester metabolises into an acid. Also methyl groups and aromatic ring can hydrolyse (arrows point towards the spot where this can occur)

 

Excretion

Excretion will be fast and mainly via the urine because of Veramoss as such and its metabolites have low molecular weight and high water solubility. There are no indications excretion via bile occurs and therefore also excretion via faeces is not expected.

 

Discussion

Veramoss is expected to be readily absorbed via the orally and via inhalation, based on physico-chemical parameters. The substance is also dermally absorbed as is shown in a dermal absorption test: 20% and it is expected based on physico-chemical properties. 

Based on this the oral absorption is at least 50%, the dermal absorption will not exceed 50% and inhalation absorption is considered to be 100% when there is exposure via the pure solid substance. This is because the inhalable fraction (< 100 um) is 70% and a small respirable fraction (< 10 um) is ca 5%. When the substance is in a liquid mixture inhalation exposure is very unlikely in view of its low vapour pressure.

 

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

 

Conclusion

Veramoss is expected to be readily absorbed via the oral and inhalation route and somewhat lower via the dermal route based physico-chemical data and data from a dermal absorption study. Using the precautionary principles for route to route extrapolation the final absorption percentages are: 50% oral absorption, 50% dermal absorption and 100% inhalation absorption.

 

References

EFSA Panel, 2012, Scientific Opinion on the safety and efficacy of primary aliphatic saturated or unsaturated alcohols/aldehydes/acids/acetals/esters with a second primary, secondary or tertiary oxygenated functional group including aliphatic lactones (chemical group 9) when used as flavourings for all animal species. EFSA Journal 10(10): 2928

 

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

 

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.

 

Yamada, T., Tanaka, Y., Hasegawa, R., Sakuratani, Y., Yamada, J., Kamata, E., Ono, A., Hirose., A., Yamazoe, Y., Mekenyan, O., Hayashi, M., 2013, A category approach to predicting the repeated-dose hepatotoxicity of allyl esters, Reg. Toxicol. Pharmacol, 65, 189-195.