Reaction mass of 2-methylbutyl acetate and pentyl acetate

Endpoint summary

Description of key information

Short description of key information on bioaccumulation potential result: 
The available data with the test substances suggest a rapid enzymatic conversion (hydrolysis) within minutes to the corresponding alcohols (and the corresponding acid metabolites to a minor extend) after incubation with fresh rat liver homogenates or after inhalation.
Short description of key information on absorption rate: 
The available data suggest a moderate absorption through human epidermis when compared with the absorption rates of other skin penetrants.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Metabolism

It was the objective of the study from Union Carbide Corporation (1993) to evaluate, if the metabolism in rat liver homogentae of the two isomeric components of the reaction mass, n-pentyl acetate and 2 -methylbutyl acetate, proceeds via hydrolysis, and, if so, to determine the Michaelis-Menten, first-order, metabolic (hydrolysis) rate constant for each isomer. For the study, the biological conditions under which these acetate esters are hydrolyzed and the characterization of the major metabolic endproducts were investigated using liver homogenate derived from adult male Fischer 344 rats. ¹⁴C-Labeled n-pentyl acetate and ¹⁴C-labeled 2-methylbutyl acetate were used and an analytical method for the analysis of ¹⁴C-n-pentyl acetate, ¹⁴C-2 -methylbutyl acetate, ¹⁴C-n-pentyl alcohol, and ¹⁴C-2-methylbutyl alcohol was developed at the testing facility, utilizing high performance liquid chromatography (HPLC) and a radioactive flow detector.

The results of preliminary testing involving the incubation of 25 mM ¹⁴C-n-pentyl acetate and ¹⁴C-2-methylbutyl acetate in 20% liver homogenates for 0-10 minutes demonstrate that essentially all of the ¹⁴C-n-pentyl acetate and ¹⁴C-2-methylbutyl acetate was converted to the corresponding alcohols. This was the confirmation that the degradation of the acetates proceeds via hydrolysis. The identities of the acetates and alcohols were confirmed by comparison with authentic ¹⁴C-radiolabeled standards, which were analyzed concurrently with the samples. Further experiments were conducted to determine that the hydrolysis of the acetates was enzymatic and to optimize experimental conditions. The hydrolysis of the acetates could be inhibited by the addition of approximately 5-10% Paraoxon (an esterase inhibitor) to the liver homogenate incubation, indicating that the hydrolysis was enzymatic.

The enzymatic nature of the hydrolysis was further demonstrated in experiments in which approximately 0.0913 mM¹⁴C-n-pentyl acetate and approximately 0.0690 mM of ¹⁴C-2-methylbutyl acetate were incubated with 0.2% liver homogenate in a 37°C water bath, and sampled for various times up to 10 minutes. The samples were then analyzed for the acetate ester and its corresponding alcohol. ¹⁴C-n-pentyl acetate, ¹⁴C-2-methylbutyl acetate, ¹⁴C-n-pentyl alcohol, and ¹⁴C-2 -methylbutyl alcohol were used to confirm the retention times of the analytes, and to measure the percent of acetate remaining and the percent of alcohol appearing in each incubation. The results indicated that there was a stochiometric relationship between the amount of acetate disappearing and the amount of the corresponding alcohol appearing in the incubation.

For determination of the metabolic rate constant for each isomer, ¹⁴C-labeled n-pentyl acetate and ¹⁴C-labeled 2-methylbutyl acetate were added to 0.2% homogenates in concentrations ranging from approximately 0.25-250 mM for approximately 1 min in a shaking, 37°C water bath. The samples were then analyzed as described above. Lineweaver-Burk plots of inverse substrate concentration (µM(-1)) versus inverse enzymatic hydrolysis velocity [(µmoles of substrate metabolized/mg protein/min)-1] were then constructed. Linear regression of these plots was used to determine the Michaelis-Menten, first-order, metabolic (hydrolysis) rate constant (Km) and the maximum velocity (Vmax) of enzymatic hydrolysis for each isomer.

The results of this study indicate that the most likely route of metabolic degradation of the two isomers in rat liver homogenates is hydrolysis, and that this breakdown is enzymatic. The calculated Km and Vmax values for n-pentyl acetate were approximately 484 µM and 0.17 µmoles/mg/min, respectively, while 2-methylbutyl acetate had calculated Km and Vmax values of approximately 70 µM and 0.09 µmoles/mg/min, respectively.

According to Dahl et al. (1987), pentyl acetate was also effectively hydrolysed in vitro by the S9 fractions obtained from nose, liver, lung, or tracheal tissue of hamsters, rats, and rabbits as determined via measurement of carboxylic acid. The liver fraction hydrolyzed pentylacetate most effectivly, followed by the ethmoturbinate. S9 fractions obtained from the trachea were almost as efficient as the ethmoturbinate from the nose in rat and hamster, and slightly less efficient in rabbits. Values for the liver were 0.25 nmol/mg S9 / min for rats and closely match the value obtained by Union Carbide, not only confirming metabolism rate, but also that hydrolysis by esterases and subsequent metabolism of the alcohol to the carboxylic acid is by far the main metabolic pathway. Dahl et al. also observed that pentyl acetate was metabolized faster then all other straight chain acetates (methylacetate through octyl acetate) and that branching lowers metabolism rates (as tested for butyl acetate). All values for pentyl acetate are presented in the following table:

Tissue	Rat	Rabbit	Hamster
Maxilloturbinates	100 +/- 8 nmol/mg S9 / min	76 +/- 13 nmol/mg S9 / min	1110 +/- 101 nmol/mg S9 / min
Ethmoturbinates	120 +/- 5 nmol/mg S9 / min	190 +/- 2 nmol/mg S9 / min	1300 +/- 33 nmol/mg S9 / min
Trachea	110 +/- 10 nmol/mg S9 / min	80 +/- 11 nmol/mg S9 / min	930 +/- 143 nmol/mg S9 / min
Lung	75 +/- 4 nmol/mg S9 / min	47 +/- 2 nmol/mg S9 / min	180 +/- 4 nmol/mg S9 / min
Liver	250 +/- 6 nmol/mg S9 / min	380 +/- 14 nmol/mg S9 / min	1100 +/- 81 nmol/mg S9 / min

Respiratory bioavailability

To determine the respiratory bioavailability of a series of alcohols and acetate esters in rats, combined evaluation of vapour uptake inhalation measurements, plethysmography and blood sampling system (for analysis of each parent chemical and its alcohol or acid metabolites) was developed (Oxo process Panel - ACC, 2004). The non-specific loss (in the absence of animals) of test substance vapours from the exposure chamber was biphasic, showing an initial more rapid loss, and generally tended to be slightly greater for the acetates than for the alcohols. The specific chamber loss rates were fairly high (up to 66% over the first hour). The additional loss of chemical from the chamber in the presence of a live rat was always substantially greater than non-specific chamber loss.

According to the results of the study the test substance present in blood after inhalative exposure (of 6 male Sprague-Dawley rats to 2000 ppm test substance vapours once for 2 hours) is rapidly converted to its alcohol metabolite (n-Pentanol and 2 - methyl butanol) as well as traces of its acid metabolites (valeric and methyl butyric acids). The alcohol metabolites exhibited the highest Cmax value, indicating a rapid conversion from the pentyl acetate parent to the alcohol, whereas the acid metabolite was near limits of detection.

Dermal absorption

The absorption of primary amyl acetate isomers from a 35:65 mix of 2-methylbutyl acetate and pentyl acetate was measured in vitro through human epidermis in a test protocol equivalent or similar to the OECD Guideline 428 (Skin Absorption: In Vitro Method; The Dow Chemical Company, 2000). The primary amyl acetate mixture was applied undiluted at a rate of 100 μl/cm²and left occluded throughout the entire exposure period (24h).

The absorption rates for both isomers of amyl acetate were essentially constant after the first 6 hours of exposure. The 2-methylbutyl acetate isomer was absorbed at a rate of 47.8 μg/cm²/h (Kp = 1.56 x 10^-4cm/h), while the pentyl acetate isomer was absorbed at a rate of 123 μg/cm²/h (Kp = 2.16 x 10^-4cm/h). These results indicate that the 2-methylbutyl acetate and pentyl acetate isomers are moderately well absorbed through human epidermis when compared with the absorption rates of other penetrants measured using this in vitro technique (Dugard et al, 1984; Dugard and Scott, 1984). The pentyl acetate isomer is absorbed through human epidermis at a faster rate than 2- methylbutyl acetate isomer.

These results are supported by a more recent in vitro study conducted with pig skin (Schenk 2018). The 2-methylbutyl acetate isomer was absorbed at a rate of 23.3 μg/cm²/h (Kp = 6.37 x 10^-4cm/h), while the pentyl acetate isomer was absorbed at a rate of 62.6 μg/cm²/h (Kp = 1.19 x 10 ^-3cm/h).