Ethylene di(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:

27 July - 29 Aug 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No hydrolysis test in saliva and gastric juice simulants.

Objective of study:

other: hydrolysis in intestinal fluid simulant

Qualifier:

according to guideline

Guideline:

other: European Food Standard Authority, Food Contact Materials, Note for Guidance (updated 30/07/2008), Annex 1 to Chapter III

Deviations:

yes

Remarks:

no hydrolysis test in saliva and gastric juice simulants

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Radiolabelling:

no

Species:

other: not specified; presumably pig in accordance with the test method used

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST DIGESTIVE SIMULANTS
INTESTINAL FLUID SIMULANT
- Description: Intestinal fluid simulant in accordance with the guideline
- Preparation: Potassium dihydrogen orthophosphate (KH2PO4) (3.41 g) was dissolved in approximately 125 mL of reverse osmosis (RO) water, in a 500 mL volumetric flask. Approximately 95 mL of 0.2 M sodium hydroxide (NaOH) was added followed by a further 200 mL of RO water. Pancreatic extract (5.00 g) was weighed into a 150 mL beaker and approximately 75 mL of RO water added gradually with stirring. The solution was then transferred to the volumetric flask containing the basified KH2PO4. Sodium taurocholate (0.25 g) was added and the mixture shaken gently. The pH of the solution was adjusted to pH 7.5 ± 0.1 using 0.2 M NaOH. The solution was diluted to volume using RO water and shaken thoroughly to mix.

Route of administration:

other: mixing

Vehicle:

unchanged (no vehicle)

Details on exposure:

- Preparation of the Test Solutions
Sample solutions were prepared in capped glass vessels at a nominal concentration of 1.5 g/L (equivalent to approximately 0.01 mol/L) in the intestinal fluid simulant.
The test solutions were split into individual vessels for each data point.
The solutions were shielded from light whilst maintained at the test temperature.

- Testing
The sample solutions were maintained at 37.0 ± 0.5 °C for a period of 1, 2 and 4 h.

Duration and frequency of treatment / exposure:

1, 2 and 4 h

Dose / conc.:

1.5 other: g/L

Remarks:

equivalent to approximately 0.01 mol/L

No. of animals per sex per dose / concentration:

triplicate determinations

Control animals:

other: for GC: blank samples from intestinal fluid simulant and samples of reference materials (parent substance and hydrolysis products), all diluted 1:10 in acetone

Details on study design:

- Principle of the Test
The test item was dissolved in the requisite test medium and the solution incubated in the dark and at 37.0 ± 0.5 °C in a water bath. The concentration of the test item was determined as a function of time, using a suitable analytical method. For decreasing concentrations, the logarithms of the concentrations were plotted against time (log10 (Ct)). For plots resulting in a straight line, the reaction is considered to be of (pseudo-) first order. The rate constant and the half-time are then calculated using the slope.

Details on dosing and sampling:

- Analysis of the Sample Solutions
Triplicate sample solutions were taken from the water bath after the specified incubation period and the pH of each solution recorded. The concentration of the sample solution was determined by gas chromatography (GC).

- Controls
Standards: Duplicate standard solutions of a mixture of test item, acetic acid and ethanediol were prepared in acetone at nominal concentration of 150 mg/L each.

Samples: An aliquot of each sample solution was diluted by a factor of 10 using acetone.

Matrix blanks: Intestinal fluid simulant diluted by a factor of 10 using acetone.

- Analysis
The standard and sample solutions were analysed by GC using the following conditions:

GC system: Agilent Technologies 6890, incorporating workstation
Column: DB-624 (60 m x 0.25 mm id x 1.4 µm film)
Oven temperature program: initial 40 °C for 2 min; rate 10 °C/min; final 80 °C for 0 min; rate 20 °C 7min; final 240 °C for 8 min
Injection mode: splitless (purge on at 0.5 min)
Flow rate: 0.75 mL/min
Injection temperature 300 °C
FID detector temperature: 250 °C
Injection volume: 1 µL
Retention time: Acetic acid ca. 9.7 min; Ethanediol ca. 11.4 min; Test item ca. 14.1 min

- Data handling
Sample solution concentration: The response factors of the standard peak areas (unit peak area per mg/L) were calculated using Equation 1:

RF = (Rstd/Cstd) [Equation 1]

Where:
RF = response factor for the standard solution
Rstd = peak area for the standard solution
Cstd = concentration for the standard solution (mg/L)

The sample solution concentration (g/L) was calculated using Equation 2

C = (Rspl/RFstd) x (D/1000) [Equation 2]

Where:
C = sample solution concentration (g/L)
Rspl = mean peak area for the sample solution
RFstd = mean response factor for the standard solutions (unit peak area per mg/L)
D = dilution factor (10)

- Degree of Hydrolysis
The decrease in concentration of test item or the degree of hydrolysis was calculated using Equation 3.

Degree of hydrolysis in percent = ((C0 – Ct)/C0) x 100 [Equation 3]

Where:
C0 = concentration at time 0
Ct = concentration at time t

- Half-life
For confirmation of first order kinetics, the data obtained in the tests were plotted as log10 (Ct) versus t.
From this plot the reaction rate constant (Kobs) was obtained from the slope, where the slope of the resultant line was calculated using linear regression analysis as per Equation 4.

Kobs = -2.303 x slope [Equation 4]

The half-life time of the reaction was calculated using Equation 5.

t1/2 = 0.693/Kobs [Equation 5]

Statistics:

Mean values of triplicate determinations were calculated.

Type:

other: degree of hydrolysis

Results:

4.37, 6.42 and 10.5% after 1, 2 and 4 h, respectively

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 25.9 h at 37 °C

Test no.:

#1

Toxicokinetic parameters:

other: rate constant = 7.42E-06 per sec

Metabolites identified:

yes

Details on metabolites:

Acetic acid concentration was determined in the initial (0 h) and in the 1, 2, and 4 h samples.
The corresponding mean concentrations were 74.9, 220, 261 and 423 mg/L at 0, 1, 2 and 4 h, respectively.
Ethanediol concentrations were not determined.
A peak in the chromatography at approximately 12.7 minutes, which was not present in the standard solutions, was observed to increase in magnitude over the period of the test. This was not identified or quantified but was considered most likely to be ethylene glycol monoacetate, a degradation product of the test item.

Table 1. Degree of hydrolysis

Sample	Sample pH (mean of 3 samples)	Mean concentration (mg/L), n=3	Log10 Concentration (mg/L)	Degree of hydrolysis (%)
Initial	7.45	1503	3.177	-
1 h	7.36	1438	3.158	4.37
2 h	7.35	1407	3.148	6.42
4 h	7.27	1345	3.129	10.5

 

Table 2. Results of linear regression

Slope	-1.16E-02
Kobs	2.67E-02 per h (7.42E-06 per sec)
t1/2	25.9 h (1.08 days)

 

Table 3. Acetic acid concentration

Sample	Mean concentration (mg/L), n=3
Initial	74.9
1 h	220
2 h	261
4 h	423

 

 

Validation

The linearity of the detector response with respect to concentration was assessed over the concentration range of 15 to 300 mg/L for both test item an acetic acid. This was satisfactory with correlation coefficients (r) of 0.999 being obtained for each analyte.

 

Discussion

It was observed that the concentration of acetic acid in the sample solutions increased with time and was most likely due to hydrolysis of the test item.

 

A peak in the chromatography at approximately 12.7 minutes, which was not present in the standard solutions, was observed to increase in magnitude over the period of the test. This was not identified or quantified but was considered most likely to be ethylene glycol monoacetate, a degradation product of the test item.

 

Conclusion

The rate constant and half-life at 37 °C of the test item in simulated intestinal fluid at pH 7.5 were determined and are shown below.

 

Table 4. Rate constant and half-life time

Rate constant (per sec)	Estimated half-life at 37 °C
7.42E-06	25.9 h

Conclusions:

Interpretation of results: bioaccumulation potential cannot be judged based on study results

Description of key information

Absorption:

Based on physico-chemical properties, a high absorption potential (oral, inhalation and dermal) is anticipated.

Distribution and accumulation:

Based on the physico-chemical properties, ethylene diacetate will be distributed within the body. It is not assumed to be accumulated as hydrolysis is anticipated to take place before absorption or during metabolism.

Metabolism:

Based on in vitro study data and physico-chemical properties, the substance will be hydrolysed to form ethylene glycol and acetic acid. An extensive subsequent metabolism of the hydrolysis products via endogenous metabolic pathways is anticipated.

Excretion:

Based on the expected metabolism, ethylene diacetate and its hydrolysis products will be mainly excreted via exhaled air as CO₂. High doses of ethylene diacetate may lead to the excretion of the metabolite oxalate via the urine.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

The hydrolysis of ethylene diacetate (CAS 111-55-7) in intestinal fluid simulant at pH 7.5 has been investigated in a study performed according to the European Food Safety Authority, Food Contact Materials, Note for Guidance (updated 30/07/2008), Annex 1 to Chapter III under GLP conditions (Harlan, 2013). No studies are available in which other parameters of the toxicokinetic behaviour, e.g., absorption and metabolism, were investigated.

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) No. 1907/2006 (REACH) and with ‘Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance’ (ECHA, 2017), assessment of the toxicokinetic behaviour of the substance ethylene diacetate was conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physico-chemical and toxicological properties, and taking into account further available information on the breakdown products of ester hydrolysis.

The substance ethylene diacetate is a diester of ethylene glycol and acetic acid and meets the definition of a mono-constituent substance based on the analytical characterisation. Ethylene diacetate is an organic liquid at 20 °C and has a molecular weight of 146.14 g/mol and a water solubility of 171.1 g/L (flask method, OECD 105). The log Pow is calculated to be 0.1 (HPLC, OECD 117) and the vapour pressure is calculated to be 22.68 Pa at 20 °C (QSAR calculation with SPARC v4.5).

Absorption

Absorption is a function of the potential of a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2017).

Oral

The smaller the molecule, the more easily it will be taken up. In general, molecular weights below 500 are favourable for oral absorption (ECHA, 2017). As the molecular weight of ethylene diacetate is 146.14 g/mol, absorption of the intact molecule in the gastrointestinal (GI) tract can be expected. Absorption after oral administration is also expected when the “Lipinski Rule of Five” ((Lipinski, 2001); Ghose (1999)) is applied to the substance.

When assessing the potential of ethylene diacetate to be absorbed in the GI tract, it has to be considered that esters will undergo hydrolysis by ubiquitously expressed carboxyl esterases (Long, 1958; Lehninger, 1970; Mattson and Nolen, 1972). The hydrolysis of ethylene diacetate was determined in an in vitro study using simulated intestinal fluid (Harlan, 2013). Following incubation of the test item (1.5 g/L) in the simulated intestinal fluid at 37 °C and pH 7.5, the degree of hydrolysis was 4.37, 6.42 and 10.5% after 1, 2 and 4 h, respectively. The rate constant for the reaction was calculated to be 7.42E-06 1/s and the half-life time was estimated to be approx. 26 h. No information on the hydrolysis of ethylene diacetate in gastric juice (pH typically 2) is available. The parent substance and the hydrolysis products are expected to be available for absorption in the GI tract. Further hydrolysis of the parent substance following absorption may occur by endogenous esterase activity, mainly in the liver. Due to the hydrolysis of the ester bond the predictions based upon the physico-chemical characteristics of the intact parent substance alone may no longer apply but also the physico-chemical characteristics of the hydrolysis products, i.e., ethylene glycol and acetic acid (ECHA, 2017).

As ethylene diacetate is highly water-soluble, the substance will readily dissolve in the GI fluids. The molecular weight of the parent substance (146.14 g/mol) suggests absorption, as described above. Furthermore, the hydrolysis products ethylene glycol and acetic acid are highly water-soluble and have low molecular weights, and can therefore dissolve in the GI fluids (acetic acid: miscible; ethylene glycol: miscible). The respective molecular weights of ethylene glycol (62.07 g/mol) and acetic acid (60.05 g/mol) also favour absorption. Due to their low molecular weights, the hydrolysis products and the intact parent substance may pass through aqueous pores or may be carried through the epithelial barrier by the bulk passage of water. Furthermore, the moderate log Pow of the substances favours absorption by passive diffusion (ethylene glycol: 1.36; acetic acid: -0.17) (ATSDR, 2010; ICPS, 2001; SCOEL, 2012; NTP, 1993).

Dermal

There are no data available on the dermal absorption or on acute dermal toxicity of ethylene diacetate. The molecular weight of ethylene diacetate (146.14 g/mol) indicates that dermal uptake of the substance is possible. The calculated octanol/water partition coefficient of 0.1 and the high water solubility suggest that the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. However, QSAR calculation using EPIwebv4.1 resulted in a Dermal Flux of 2.04E-1 mg/cm² per h and QSAR calculation using DERMWIN showed a high dermal absorption potential with a permeability constant of 4.42E-04 cm/h of ethylene diacetate. Available data on acute dermal toxicity of the structurally related substance propane-1,2-diyl diacetate (CAS 623-84-7) did not show signs of systemic toxicity, resulting in an LD50 value > 2000 mg/kg bw (Notox, 1986a). In addition, irritation and sensitisation studies with either ethylene diacetate or the structurally related substance propane-1,2-diyl diacetate showed no irritating effects to skin and eyes and no signs of systemic toxicity were determined in respective studies (Carpenter, 1946; Mellon, 1965; Hazleton, 1984; Notox, 1986b). Based on the physico-chemical properties of ethylene diacetate, the lack of systemic dermal toxicity observed can be attributed to a low dermal toxicity potential of the substance.

Overall, taking into account the physico-chemical properties of ethylene diacetate and the QSAR calculation, the dermal absorption potential of the test substance is predicted to be high.

Inhalation

Ethylene diacetate has a very low vapour pressure of 22.68 Pa at 20 °C, which indicates the substance will show low volatility. Therefore, under normal use and handling conditions, inhalation exposure and the availability for respiratory absorption of the substance in the form of vapours, gases, or mists is insignificant. However, the substance may be available for respiratory absorption after inhalation of aerosols, if the formulated substance is sprayed. In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract (ECHA, 2017). Due to the high hydrophilicity of ethylene diacetate, deposition in the mucus of the upper respiratory tract is possible. Due to the moderate log Pow value of ethylene diacetate and the predicted hydrolysis products ethylene glycol and acetic acid, direct absorption across the respiratory tract epithelium by passive diffusion is favoured. Absorption of deposited material is predicted to be high, due to the low molecular weight and the moderate log Pow values of the parent substance and the hydrolysis products, as discussed in the oral absorption section above. Data from acute studies via inhalation and oral route from the structurally related substance propane-1,2-diyl diacetate did not show systemic toxicity. However, this lack of short-term systemic toxicity of the analogue substance indicates a low toxic potential of the test substance and the hydrolysis products, rather than a lack of absorption via the inhalation route.

In conclusion, based on the physico-chemical properties of ethylene diacetate and data on acute inhalation toxicity of the structurally related substance propane-1,2-diyl diacetate absorption via the inhalation route is expected to be high.

Distribution and accumulation

Distribution of a compound within the body depends on the physico-chemical properties of the substance; especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution (ECHA, 2017). As the parent compound ethylene diacetate will be hydrolysed to a substantial extent before absorption or in the liver following absorption, the distribution of intact ethylene diacetate and of the hydrolysis products is relevant. Ethylene diacetate, ethylene glycol and acetic acid can be distributed within the body. Both, the parent substance and the hydrolysis products, ethylene glycol and acetic acid, have low molecular weights and high water solubility. Based on the physico-chemical properties, ethylene diacetate, ethylene glycol and acetic acid will be distributed within the body (ATSDR, 2010; IPCS, 2001; Yalkowsky, 2003; Riddick, 1986). Substances with high water solubility do not have the potential to accumulate in adipose tissue due to their low log Pow. In addition, the intact parent compound is not likely to be accumulated as hydrolysis is predicted to take place before absorption or during metabolism (see below).

Metabolism

The metabolism of ethylene diacetate is expected to occur initially via enzymatic hydrolysis of the ester by ubiquitous expressed esterases before absorption. In in vivo studies in rats with esters containing ethylene glycol, it was shown that they are rapidly hydrolysed by ubiquitously expressed esterases and almost completely absorbed (Mattson and Volpenhein, 1968; Mattson and Nolen 1972). The fraction of ester absorbed unchanged will undergo enzymatic hydrolysis by ubiquitous esterases, primarily in the liver (Fukami and Yokoi, 2012). Furthermore, in vivo studies with the structurally related ester triacetin showed that during intravenous administration in dogs, the majority of infused triacetin undergoes intravascular hydrolysis, and the majority of the resulting acetate will be oxidised (Bleiberg, 1993). In a simulation of intestinal metabolism of ethylene diacetate performed using the OECD QSAR Toolbox (version 4.2, 2018), metabolites including ethylene monoacetate, acetic acid and oxidised ethylene glycol derivatives (e.g. glyoxylic acid) were predicted, supporting the metabolism pathway of a stepwise ester hydrolysis. Similarly, liver metabolism simulation resulted in several metabolites including ethylene monoacetate and acetic acid. Following hydrolysis, absorption and distribution of the alcohol component, ethylene glycol will be metabolised primarily in the liver. Ethylene glycol has been shown to be oxidised in experimental animals and in humans in successive steps, first to glycoaldehyde, catalysed by alcohol dehydrogenase, then to glycolic acid, glyoxylic acid, and oxalic acid. Glyoxylic acid is metabolised in intermediary metabolism to malate, formate, and glycine. Ethylene glycol, glycolic acid, calcium oxalate, glycine and its conjugate, hippurate are excreted in urine. The metabolites of ethylene glycol that have been typically detected are CO₂, glycolic acid, and oxalic acid (WHO, 2002). The predicted metabolite ethylene glycol is classified as acutely toxic (oral), category 4, according to Regulation (EC) No. 1272/2008, Annex VI (CLP). The effects observed in laboratory animals and humans are due primarily to the actions of one or more of its metabolites, rather than to the parent compound ethylene glycol (WHO, 2002). The available animal data on the intact ester, ethylene diacetate, showed that no acute oral toxicity was observed in rats up to and including the highest applied doses of 6860 mg/kg bw. It is therefore considered unlikely that acute toxicity will be caused by the metabolism of the ester to the primary metabolite ethylene glycol and by additional steps to the potentially toxic metabolites of ethylene glycol. The second product of hydrolysis, acetic acid and the respective acetate ion are normally-occurring metabolites in catabolism or in anabolic synthesis, e.g. in the formation of glycogen, cholesterol synthesis and degradation of fatty acids (SCOEL, 2012). Available genotoxicity data from the parent substance ethylene diacetate and the structurally related substance of propane-1,2-diyl diacetate did not show any genotoxic properties. All available studies were consistently negative and therefore no genotoxic reactivity of ethylene diacetate and the structurally related substance propane-1,2-diyl diacetate under the test conditions is indicated.

Excretion

Based on the metabolism described in the paragraphs above, ethylene diacetate and its hydrolysis products will be metabolised in the body to a high extent. Acetic acid will be metabolised in the citric acid cycle and ultimately primarily excreted via exhaled air as CO₂ (Lehninger, 1970; Stryer, 1994). As ethylene glycol will be highly metabolised as well, the primary route of excretion will be via exhaled air as CO₂ and as parent compound and glycolic acid in the urine. Higher doses of ethylene glycol lead to the excretion of the metabolite oxalate via the urine (ATSDR, 2010).

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