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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
No details about environmental conditions but similarities with the study in male rats are presumed.
Objective of study:
toxicokinetics
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
not specified
Remarks:
GLP statements are usually not mentioned in publications in journals
Radiolabelling:
yes
Remarks:
[1,2-14C]-ethylene glycol from Sigma Chemicals, specific activity 3.6 mCi/mmol radiochemical, purity: 99.2% (analysed by GC)
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Source: Harlan Sprague-Dawley, Inc., Indianapolis, USA
age: 10-11 weeks (180-250 g bw)
acclimatisation: 2 days
Route of administration:
other: oral, dermal or intravenous (i.v.)
Vehicle:
other: see details
Details on exposure:
Oral
Gavage; vehicle water, rats were fasted for 15-16 h prior to application; concentration in vehicle 0.5, 20, 30, 40, 50%, respectively; total volume applied: 2 ml/kg bw; analytical control of dose via liquid scintillation spectrometry
Dermal
Preparation of test site not specified; dose 10 or 1000 mg/kg bw undiluted or 1000 mg/kg bw 50% w/w water solution (15-25 μCi/animal); volume applied: 2 ml/kg bw applied to 1 cm² in the interscapular region of the back by special syringe method; exposure period: 6 hours under occlusive covering; then occlusive covering was removed and any unabsorbed test substance was washed from the dose site with water-wettened, cotton-tipped applicators; replacement with fresh covering for the total 96-hour test period
i.v.
Application via indwelling jugular cannula which was surgically implanted 48 hours prior to dose administration; target dose 10 or 1000 mg/kg bw, corresponding to 5 μCi/animal; vehicle physiological saline; total volume applied: 2 ml/kg bw



Duration and frequency of treatment / exposure:
once
Remarks:
Doses / Concentrations:
Oral dose: 10, 400, 600, 800, 1000 mg/kg bw (10-15 μCi/ animal)
Dermal: 10 or 1000 mg/kg bw undiluted or 1000 mg/kg bw 50% w/w water solution
i.v.: 10 or 1000 mg/kg bw
No. of animals per sex per dose:
4/dose/route
Control animals:
no
Details on study design:
Dose selection rationale: NOAEL/LOAEL in repeated dose toxicity studies
Details on dosing and sampling:
Sampling time
Blood (via jugular cannula): 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, and 96 h after dosing.
Exceptions: Additional blood samples at 5 and 15 min post-dosing for the intravenous application. For the 400, 600 and 800 mg/kg bw gavage groups blood sampling only at 24, 36, 48, 72, and 96 h after dosing.
Urine and faeces samples: 12, 24, 36, 48, 72, 96 h post dosing
Expired 14CO2: air flow through metabolism cages ca. 500 ml/min; expired CO2 trapped in 12-24-hour intervals using solutions of 2-methoxymethanol:ethanolamine (7:3)

Samples for distribution of 14C-activity
Urine, faeces, plasma, 14CO2-trap solution sampled at indicated timepoints.
Total exsanguination via cardiac puncture 96 hours post-dosing.
Faeces and tissues were prepared as 33% water homogenates.
Radioactivity of these samples was analysed by liquid scintillation spectrometry.
Skin sample for dermal study: dosed area together with the skin around the periphery (approximately 1 cm further); sample pulverized at liquid nitrogen temperature, radioactivity of aliquots counted directly as suspension by liquid scintillation spectrometry.

Analysis of unmetabolized ethylene glycol
Plasma samples (timepoints see above) remaining after radioanalysis were pooled in equal volumes and derivatized with phenylboronic acid (PBA). PBA-derivatized ethylene glycol was analysed using capillary GC with a Mass Selective Detector and was identified using Selective Ion Monitoring techniques. Limit of quantitation: ca. 1 ng/µl

Profiles of ethylene glycol metabolites in plasma
Separate groups to obtain larger plasma volumes: 2 rats/interval
Studied doses: 10 and 1000 mg/kg bw via gavage
Sampling times: exsanguination via cardiac puncture at 2, 4, 6, 8, 10 and 12 h after administration
Preparation of plasma samples via ultrafiltration using Centricon-10 concentrators
Analysis of plasma samples: HPLC with ion exchange column, detection with in-line radioactivity flow monitor and differential refractive index detector; analyzed metabolites: ethylene glycol, glycolic acid (glycolate), glycolaldehyde, glyoxylic acid (glyoxylate), oxalic acid (oxalate); glyoxalate and glyoxal are not distinguishable in this HPLC system
Limit of quantitation: 33 ng/100 µl of injected plasma filtrate

Profiles of ethylene glycol metabolites in urine
Selected urine samples from 4 rats/dose were analysed for metabolites: selection criterium was the appearance of 14C-activity in total urine.
Analyzed samples, application mode and dose groups:
Analysis of urine samples: HPLC with ion exchange column, detection with in-line radioactivity flow monitor and differential refractive index detector
Analyzed metabolites: ethylene glycol, glycolic acid (glycolate), glycolaldehyde, glyoxylic acid (glyoxylate), glyoxal, oxalic acid (oxalate)
Limit of quantitation: ca. 1 µg ethylene glycole/ml urine; ca. 2 µg metbolite/ml urine

Pharmacokinetic evaluations
Pharmacokinetic data analysis was based on semilogarithmic plots for concentrations of both total radioactivity and unchanged ethylene glycol in plasma versus time.
Analysis of elimination and transfer of 14C- ethylene glycol between compartments: mean plasma 14C concentrations were used initially to fit one-, two- or three-exponential equations. Both were assumed to be first-order processes.
Plasma concentration-time data:
14C-concentrations after intravenous and dermal application, and ethylene glycol concentration after oral administration: described by a biexponential equation of the form: Ct = AeExp-alpha t + Be Exp-beta t.
14C-concentrations after oral administration described by a triexponential equation and ethylene glycol concentration after intravenous administration described by a monoexponential equation.
Parameters estimated based on mean plasma concentration values:
beta: rate constant for terminal disposition
AUC: area under the curve
Vdss: apparent volume of distribution at steady state
Cl oral: clearance of ethyleneglycol after oral doslng
Cl total = [dose : AUC] x 1000 µg/mg
MRT: mean residence time
t½: half-times of elimination
U EG: % administered dose excreted unchanged via urine (0-24 h)
F: fraction of the parent dose absorbed, or its bioavailable dose
Cmax: max plasma concentration
tmax: time to maxlmum concentration



Statistics:
means and standart deviation
Type:
other: see detailed text below
Details on absorption:
see detailed text below
Details on distribution in tissues:
see detailed text below
Details on excretion:
see detailed text below
Metabolites identified:
yes
Details on metabolites:
see detailed text below
Bioaccessibility testing results:
see detailed text below

Absorption

Oral route

Results presented in Table 1 have shown that bolus gavage doses of 10 or 1000 mg/kg bw ethylene glycol (EG) were rapidly and almost completely absorbed in female rats, with a bioavailable fraction of 92%. Uptake and elimination of EG from plasma was biexponential; elimination rates and t1/2 beta values were of similar duration between dose levels. Overall, a linear pharmacokinetic relationship for parent EG was apparent for both oral dose levels. Data on oral doses of 400, 600 or 800 mg/kg bw (not shown in Table 1) suggested that plasma 14C elimination is independent of dose in this range.

Dermal route

In contrast to the oral route, the absorption of cutaneously applied EG is comparatively slow in female rats (see Table 1). EG has a bioavailability of only 25%. Total absorption of radioactivity varied between 26-32% (see Table 2).

Total recoveries

Recoveries of total radioactivity were within 83 to 92% with exception of the 10 mg/kg bw dermal application with a reduced recovery of 42% only (see Table 2).

Tissue distribution of 14C-activity (see Table 2 and 3)

Intravenous and oral route (10, 1000 mg/kg bw):

Total tissue recovery of 14C accounted to 5.3 and 5.7% at the low dose. At the high dose there was a nearly twofold decrease to 3.3 and 2.2%. Concentrations of 14C in individual tissues showed a dose-dependence being about two orders of magnitude lower at the low dose compared to high dose tissue values. Tissue/plasma ratios > 1.0 were calculated for liver, kidney, and lung, indicating a greater presence of 14C in these tissues than would be accounted by plasma perfusion alone.

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol):

The distribution of 14C to tissues showed similar disposition profiles to those observed after i.v. and p.o. dosing. Tissue/plasma ratios > 1.0 were calculated for: liver, kidney, and pelt at 10 mg/kg bw, for liver, kidney, lung and pelt at 1000 mg/kg (undiluted), and for liver and pelt at 1000 mg/kg bw (aqueous dilution), indicating a greater presence of 14C in these tissues than would be accounted by plasma perfusion alone.

Excretion (see Table 2, 4 and 5)

Total excretion of radioactivity

14C-activity was recovered from urine, faeces and as 14CO2 in exhaled air in percentages varying for different application routes and doses.

Intravenous and oral route (10, 1000 mg/kg bw): 10 mg/kg bw: major excretion route: exhalation 44-48%; urine: 25%; faeces: 2.0-2.8%. 1000 mg/kg bw (values for i.v. and p.o. route, respectively): major excretion route: urine 45%, 35%; exhalation 29%, 28%; faeces: 5.4%, 4.4%.

Oral route (400, 600, 800 mg/kg bw): At 400 mg/kg bw excretion via exhalation and urine accounted to approximately equal amounts. In the dose range up to 800 mg/kg bw there was a dose-dependent increase of urinary excretion being accompanied by a decrease of 14CO2 exhalation. This points to saturation of oxidative metabolic pathways.

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol): 10 mg/kg bw: major excretion route: exhalation 13.1%; urine 8.2%; faeces 1.1%. 1000 mg/kg bw: exhalation 11.4%; urine 7.6%; faeces 1.4%. Dermal route (1000 mg/kg bw, undiluted ethylene glycol): major excretion route: exhalation 9.3%; urine 4.4%; faeces 0.5%.

Time course of excretion of radioactivity and metabolites

Intravenous and oral route (10, 1000 mg/kg bw): Elimination profiles for total radioactivity were similar in both time-course and amount of the dose excreted for both application routes and for each dosage, indicating that the majority of the oral doses were absorbed. For both routes, at the low dose excretion of radioactivity was mainly via exhalation as 14CO2. In contrast, at the high dose urinary excretion was the dominant route. Urinary excretion: majority of 14C excreted within 12 h, urinary excretion virtually complete within 24-36 h post-dosing. At the high 1000 mg/kg-dose a higher percentage was excreted via urine (i.v. 45%, p.o. 35%) than after the low 10 mg/kg-dose (25%).

10 mg/kg bw: Urinanalysis of metabolites yielded a similar pattern for the low dose for both application routes with unchanged ethylene glycol accounting to ca. 93% of total 14C during the first 12 hours. Glycolic acid (ca. 5%) and oxalic acid (<1%) were found as minor metabolites. 1000 mg/kg bw: During the 12-24 h interval excretion of unchanged ethylene glycol accounted to 53% (i.v.) and 65% (p.o.) besides glycolic acid (46% and 35%). Oxalic acid was found only in minor amounts (<1%).

Exhalation of 14CO2: There was still a slow increase of cumulative exhaled 14CO2 at the end of the 96-h observation period. Within this interval the majority of 14CO2 was exhaled within the first 24 h. Overall levels were inversely related to dose (44-48% at the low dose, 28% at the high dose).

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol): Elimination profiles for total radioactivity for both doses showed a continuous increase of excretion during the 96-h observation period up to a final excretion of 8-10% of the dose via urine and of 13% via exhalation. Total excretion was lower than after i.v. or p.o. application. However, taking the low dermal penetration into account, excreted relative amounts were comparable with excretion percentages after i.v. or p.o. dosing. In the urine fractions unchanged ethylene glycol was detected as the major component (87-100%). Dermal route (1000 mg/kg bw, 50 % aqueous dilution of ethylene glycol): Measurable excretion of 14C was retarded until 24 h post-dosing. There was a continuous increase during the 96-h observation period until final levels of 5% in urine and 9% in exhaled air. Radioactivity in urine accounted to 100% to unchanged ethylene glycol.

Conclusions:
No bioaccumulation potential based on study results.
Ethylene glycol is very rapidly and almost completely absorbed after oral doses but dermally-applied EG was slowly and rather poorly absorbed; oxidative metabolite pathways appeared to be saturated at high oral doses; linear pharmacokinetic relationship was apparent for the oral route.
Executive summary:

This study was comparable to OECD guideline 417. 14C-ethylene glycol was administered to female Sprague-Dawley rats (n=4 per group) intravenously, orally or dermally in single doses ranging from 10 – 1000 mg/kg bw. Pharmacokinetic investigations of the fate of ethylene glycol included absorption from the gastrointestinal tract, skin penetration, tissue distribution, identification of metabolites in urine, and pathways and kinetics of excretion.

Results have shown that gavage doses of 10 or 1000 mg/kg bw ethylene glycol (EG) were rapidly and almost completely absorbed in female rats, with a bioavailable fraction of 92%. Uptake and elimination of EG from plasma was biexponential; elimination rates and t1/2 beta values were of similar duration between dose levels. Overall, a linear pharmacokinetic relationship for parent EG was apparent. In contrast to the oral route, the absorption of cutaneously applied EG is comparatively slow in female rats. EG has a bioavailability of only 25%. Total dermal absorption of radioactivity varied between 26-32%. Relative recoveries of 14C in tissues and carcass were higher at the 10 mg/kg bw p.o. and i.v. dose in comparison to the 1000 mg/kg bw dose, whereas individual 14C concentrations in tissues increased dose-dependently. Storage in tissue accounted to total amounts of ca. 2-6 % of the dose after 96 hours. Extensive metabolism via oxidative pathways is indicated by the high amounts of 45-49 % of the dose exhaled as 14CO2 at 10 mg/kg. Expiration of 14CO2 diminishes to 30 % as the dose increases to 1000 mg/kg bw. Parallel to the decrease of excretion via exhalation urinary excretion increases from 25% to 36-46% of the dose. Urinary excretion profiles show unchanged ethylene glycol as the main source of 14C besides glycolate as the major urinary metabolite and minor amounts of oxalate (<1%). Only minor amounts of 14C are both excreted in the faeces (ca. 2-5%) and remain in the carcass (ca. 5-9 %).

Conclusions: Ethylene glycol is very rapidly and almost completely absorbed after oral doses but dermally-applied EG was slowly and rather poorly absorbed; oxidative metabolite pathways appeared to be saturated at high oral doses; linear pharmacokinetic relationship was apparent for the oral route.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
no details about environmental conditions but similarities with the study in male rats are presumed
Objective of study:
toxicokinetics
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
not specified
Remarks:
GLP statements usually are not mentioned in publications in journals
Radiolabelling:
yes
Remarks:
[1,2-14C]-ethylene glycol from Sigma Chemicals, specific activity 5.8-10.1 mCi/mmol, purity > 98 % (analysed by GC)
Species:
mouse
Strain:
CD-1
Sex:
female
Details on test animals and environmental conditions:
5-6 weeks old, 15-25 g bw, obtained from Charles River Laboratories, lnc.
acclimatisation: 2 days
Route of administration:
other: oral, dermal or intravenous (i.v.)
Vehicle:
other: see details
Details on exposure:
Oral
Gavage; 16 h fasting prior to gavage, vehicle water; concentration in vehicle 0.5, 5, 10, 20, 50%, respectively; total volume applied: 2 ml/kg bw; analytical control of dose via liquid scintillation spectrometry
Dermal
skin clipped over a 2 cm x 3 cm area an the dorsal surface; dose 100 or 1000 mg/kg bw undiluted or 1000 mg/kg bw 50% w/w water solution (15-25 μCi/animal); volume applied: 2 ml/kg bw applied to 1 cm² in the interscapular region of the back by special syringe method; exposure period: 6 hours under occlusive covering
i.v.
dose 10 or 1000 mg/kg bw, corresponding to 5 μCi/animal; vehicle physiological saline; total volume applied: 2 ml/kg bw



Duration and frequency of treatment / exposure:
once
Remarks:
Doses / Concentrations:
Oral dose: 10, 100, 200, 400, 1000 mg/kg bw (10-15 μCi/ animal)
Dermal: 100 or 1000 mg/kg bw undiluted or 1000 mg/kg bw 50% w/w water solution
i.v.: 10 or 1000 mg/kg bw
No. of animals per sex per dose:
4/dose/route
Control animals:
no
Details on study design:
Dose selection rationale: NOAEL/LOAEL in repeated dose toxicity studies
Details on dosing and sampling:
Sampling time
Urine and faeces samples: 12, 24, 36, 48, 72, 96 h post dosing
Expired 14CO2: air flow through metabolism cages ca. 500 ml/min; expired CO2 trapped in 12-24-hour intervals using silica gel traps
Volatile organics sampled trapped in 12-24-hour intervals using silica gel traps


Samples for distribution of 14C-activity
Urine, faeces, plasma, 14CO2-gel trap sampled at indicated timepoints.
Total exsanguination via cardiac puncture 96 hours post-dosing.
Faeces and tissues were prepared as 33% water homogenates.
Radioactivity of these samples was analysed by high temperature combustion (900° C).
Skin sample for dermal study: dosed area together with the skin around the periphery (approximately 1 cm further); sample pulverized at liquid nitrogen temperature, radioactivity of aliquots counted directly as suspension by liquid scintillation spectrometry.

Profiles of ethylene glycol metabolites in urine
Selected urine samples from 4 mice/dose were analysed for metabolites: selection criterium was the appearance of 14C-activity in total urine.
Analyzed samples, application mode and dose groups:
Intravenous: 10, 1000 mg/kg bw
Peroral (via gavage): 10, 1000 mg/kg bw
Percutaneous: undiluted 10, 1000 mg/kg bw; 50% aqueous solution 1000 mg/kg bw
Sampling times: 0-12 h and 12-24 h intervals
Analysis of urine samples: HPLC with ion exchange column, detection with in-line radioactivity flow monitor and differential refractive index detector
Analyzed metabolites: ethylene glycol, glycolic acid (glycolate), glycolaldehyde, glyoxylic acid (glyoxylate), glyoxal, oxalic acid (oxalate)
Limit of quantitation: ca. 1 µg ethylene glycole/ml urine; ca. 2 µg metbolite/ml urine


Statistics:
means and standart deviation
Type:
other: see detailed text below
Details on absorption:
see detailed text below
Details on distribution in tissues:
see detailed text below
Details on excretion:
see detailed text below
Metabolites identified:
yes
Details on metabolites:
see detailed text below
Bioaccessibility testing results:
see detailed text below

Absorption

Oral route:

Conclusions on absorption from the gastrointestinal tract are possible from the comparison of the disposition of 14C-activity in tissues and excreta (see Table 1) after intravenous and oral application. Similar percentages of 14C-activity in faeces, urine, and exhaled air and similar tissue recoveries point to a virtually complete absorption from the gastrointestinal tract.

Dermal route:

Apparent absorbed doses are available from the recoveries of 14C-activity from excreta, tissues and carcass. Dermal absorption accounted to 77-84% for undiluted ethylene glycol and 60% for the aqueous dilution.

Total recoveries

Recoveries of total radioactivity were within 85 to 102%. (see Table 1).

Tissue distribution of 14C-activity (see Table 2 and 3)

Intravenous and oral route:

Total tissue recovery of 14C accounted to 2.6 and 3.6% at the low dose. At the high dose there was a nearly fourfold decrease to 0.7-0.8%. Concentrations of 14C in individual tissues showed a dose-dependence being about two orders of magnitude lower at the low dose compared to high dose tissue values. Tissue/plasma ratios > 2.0 were calculated for liver, kidney, and lung, indicating a greater presence of 14C in these tissues than would be accounted by plasma perfusion alone.

Dermal route:

The distribution of 14C to tissues showed similar disposition profiles to those observed after i.v. and p.o. dosing.

Tissue/plasma ratios > 2.0 were calculated for: liver, kidney, fat, lung and carcass/pelt at 100 mg/kg bw and at 1000 mg/kg bw (aqueous dilution), and for liver, kidney, and carcass/pelt at 1000 mg/kg bw (undiluted), indicating a greater presence of 14C in these tissues than would be accounted by plasma perfusion alone.

Excretion (see Table 1, 3, and 4)

Total excretion of radioactivity

14C-activity was recovered from urine, faeces and as exhaled 14CO2 and volatile organics in percentages varying for different application routes and doses.

The percentages of volatile organics were distinctly higher after dermal application (ca. 34% for undiluted, 21% for diluted ethylene glycol samples) in comparison with i.v. or oral uptake with organic volatiles accounting to 3-5%. This increased volatile radioactivity fraction possibly represents ethylene glycol that evaporated from the backs of the mice over the 96 h observation period.

Intravenous and oral route (10, 1000 mg/kg bw), respectively:

10 mg/kg bw: major excretion route: exhalation 50 and 55%; urine: 24%; faeces: 4.6 and 6.6%; organic volatiles 3.1%

1000 mg/kg bw (values for i.v. and p.o. route): major excretion route: urine 47%, 56%; exhalation 24%, 22%; faeces: 10.3%, 3.7%; organic volatiles 4.9, 4.2%

Oral route (100, 200, 400 mg/kg bw):

At 100 mg/kg bw excretion via exhalation and urine accounted to approximately equal amounts. In the dose range up to 400 mg/kg bw there was a dose-dependent increase of urinary excretion being accompanied by a decrease of 14CO2 exhalation.

Dermal route (100, 1000 mg/kg bw, undiluted ethylene glycol):

100 mg/kg bw: major excretion route: organic volatiles 34.%; exhalation 10.4%; urine 8.2%; faeces 1.1%

1000 mg/kg bw: organic volatiles 33.%; exhalation 15.9%; urine 7.3%; faeces 7.5%.

Dermal route (1000 mg/kg bw, undiluted ethylene glycol):

major excretion route: organic volatiles 21 %; exhalation 10.4%; urine 5.4%; faeces 6.4%.

Time course of excretion of radioactivity and metabolites

Intravenous and oral route (10, 1000 mg/kg bw):

Elimination profiles for total radioactivity were similar in both time-course and amount of the dose excreted for both application routes and for each dosage, indicating that the majority of the oral doses were absorbed. For both routes, at the low dose excretion of radioactivity was mainly via exhalation as 14CO2. In contrast, at the high dose urinary excretion was the dominant route.

Urinary excretion:

majority of 14C excreted within 12 h, urinary excretion virtually complete within 24-36 h post-dosing. At the high 1000 mg/kg-dose a higher percentage was excreted via urine (i.v. 46%, p.o. 54%) than after the low 10 mg/kg-dose (24%).

Intravenous route:

10 mg/kg bw: Unchanged ethylene glycol levels remained at approximately 85% during the 96 h observation period. About 10-15% of the dose was voided as glycolate.

1000 mg/kg bw: Unchanged ethylene glycol levels decreased with time after dosing while glycolate excretion increased.

Peroral route:

10 mg/kg bw: The level of unchanged ethylene glycol decreased steadily with time over the first 48 hours so that ethylene glycol was not detectable in urine at the 72 h-timepoint. During the initial 36 h post-dosing ca. 65% of urinary excretion accounted to the parent compound and ca. 35% to glycolate. Glycolate rose steadily thereafter.

1000 mg/kg bw: Unchanged ethylene glycol levels decreased with time after dosing while glycolate excretion increased.

Exhalation of 14CO2:

There was still a slow increase of cumulative exhaled 14CO2 at the end of the 96-h observation period. Within this interval the majority of 14CO2 was exhaled within the first 24 h. Overall levels were inversely related to dose (50-55% at the low dose, 22-24% at the high dose).

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol):

Elimination profiles for total radioactivity for both doses showed a continuous increase of excretion during the 96-h observation period up to a final excretion of 6 and 12% of the dose via urine and of 10 and 16% via exhalation. In the urine fractions unchanged ethylene glycol was detected besides glycolate as metabolite.

Dermal route (1000 mg/kg bw, 50 % aqueous dilution of ethylene glycol):

Measurable excretion of 14C was retarded until 24 h post-dosing. There was a continuous increase during the 96-h observation period until final levels of 5% in urine and 10% in exhaled air. Radioactivity in urine accounted to unchanged ethylene glycol and glycolate.

Conclusions:
No bioaccumulation potential based on study results.
In mice, ethylene glycol is completely and rapidly absorbed after oral application, absorption via skin is lower and accounted to ca. 40-50% of applied dose.
Executive summary:

The applied test method was comparable to OECD guideline 417.

14C- ethylene glycol was administered to female mice intravenously, orally or dermally in single doses ranging from 10 – 1000 mg/kg bw. Pharmacokinetic investigations of the fate of ethylene glycol included absorption from the gastrointestinal tract, skin penetration, tissue distribution, identification of metabolites in urine, and pathways and kinetics of excretion.

Depending on the route of administration differences in the amount of absorbed 14C ethylene glycol are found. Similar disposition of 14C concentrations in excreta and in tissues after p.o. and i.v. application gives evidence of virtually complete absorption from the gastrointestinal tract. Absorption of ethylene glycol via mouse skin is lower. Apparent absorbed doses are available from the recoveries of 14C-activity from excreta and tissues. Dermal absorption accounted to ca. 40-50% when the volatile organic fraction is considered as evaporation of ethylen glycol from the skin and is substracted from the apparent absorption.

Relative recoveries of 14C in tissues and carcass were higher at the 10 mg/kg bw p.o. and i.v. dose in comparison to the 1000 mg/kg bw dose, whereas individual 14C concentrations in tissues increased dose-dependently. Storage in tissue is low after 96 hours with total amounts of ca. 3 and <1% of the dose at 10 and 1000 mg/kg bw.

Extensive metabolism via oxidative pathways is indicated by the high amounts of 50-55 % of the dose exhaled as 14CO2 at 10 mg/kg bw. Expiration of 14CO2 diminishes to ca. 23 % as the dose increases to 1000 mg/kg bw. Parallel to the decrease of excretion via exhalation urinary excretion increases from 23% to 47-56% of the dose.

Urinary excretion profiles show unchanged ethylene glycol as the main source of 14C besides glycolate as detectable urinary metabolite. As the dose increases excretion of glycolate exceeds that of unchanged compound. Smaller amounts of 14C are both excreted in the faeces (ca. 4-10%) and remain in the carcass (ca. 3-15 %).

Conclusion: In mice, ethylene glycol is completely and rapidly absorbed after oral application, absorption via skin is lower and accounted to ca. 40-50% of applied dose.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Objective of study:
toxicokinetics
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
not specified
Remarks:
GLP statements are usually not mentioned in publications in journals
Radiolabelling:
yes
Remarks:
[1,2-14C]-ethylene glycol from Sigma Chemicals, specific activity 3.6 mCi/mmol radiochemical, purity: 99.2% (analysed by GC)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Source: Harlan Sprague-Dawley, Inc., Indianapolis, USA
age: 10-11 weeks (275-350 g bw)
acclimatisation: 2 days
conditions: air humidity 40-70% and temperature 64-79°F, with a 12-h photoperiod
Route of administration:
other: oral, dermal or intravenous (i.v.)
Vehicle:
other: see details
Details on exposure:
Oral
Gavage; vehicle water, concentration in vehicle 0.5, 20, 30, 40, 50%, respectively; total volume applied: 2 ml/kg bw; analytical control of dose via liquid scintillation spectrometry
Dermal
Preparation of test site not specified; dose 10 or 1000 mg/kg bw undiluted or 1000 mg/kg bw 50% w/w water solution (15-25 µCi/0.2 ml tracer per 200 g rats); volume applied: 2 ml/kg bw applied to 1 cm² in the interscapular region of the back by special syringe method; exposure period: 6 hours under occlusive covering; then occlusive covering was removed and any unabsorbed test substance was washed from the dose site with water-wettened, cotton-tipped applicators; replacement with fresh covering for the total 96-hour test period
i.v.
Application via indwelling jugular cannula which was surgically implanted 48 hours prior to dose administration; target dose 10 or 1000 mg/kg bw, corresponding to 5-10 µCi/rat; vehicle physiological saline; voncentration in vehicle: 5 or 500 mg/ml; total volume applied: 2 ml/kg bw



Duration and frequency of treatment / exposure:
once
Remarks:
Doses / Concentrations:
Oral dose: 10, 400, 600, 800, 1000 mg/kg bw
Dermal: 10 or 1000 mg/kg bw undiluted or 1000 mg/kg bw 50% w/w water solution
i.v.: 10 or 1000 mg/kg bw
No. of animals per sex per dose:
6/dose/route
Control animals:
no
Details on study design:
Dose selection rationale: NOAEL/LOAEL in repeated dose toxicity studies
Details on dosing and sampling:
Sampling time
Blood (via jugular cannula): 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, and 96 h after dosing.
Exceptions: Additional blood samples at 5 and 15 min post-dosing for the intravenous application. For the 400, 600 and 800 mg/kg bw gavage groups blood sampling only at 24, 36, 48, 72, and 96 h after dosing.
Urine and faeces samples: 12, 24, 36, 48, 72, 96 h post dosing
Expired 14CO2: air flow through metabolism cages ca. 500 ml/min; expired CO2 trapped in 12-24-hour intervals using solutions of 2-methoxymethanol:ethanolamine (7:3)

Samples for distribution of 14C-activity
Urine, faeces, plasma, 14CO2-trap solution sampled at indicated timepoints.
Total exsanguination via cardiac puncture 96 hours post-dosing.
Faeces and tissues were prepared as 33% water homogenates.
Radioactivity of these samples was analysed by high temperature combustion at 900°C.
Skin sample for dermal study: dosed area together with the skin around the periphery (approximately 1 cm further); sample pulverized at liquid nitrogen temperature, radioactivity of aliquots counted directly as suspension by liquid scintillation spectrometry.

Analysis of unmetabolized ethylene glycol
Plasma samples (timepoints see above) remaining after radioanalysis were pooled in equal volumes and derivatized with phenylboronic acid (PBA). PBA-derivatized ethylene glycol was analysed using capillary GC with a Mass Selective Detector and was identified using Selective Ion Monitoring techniques. Limit of quantitation: ca. 1 ng/µl

Profiles of ethylene glycol metabolites in plasma
Separate groups to obtain larger plasma volumes: 2 rats/interval
Studied doses: 10, 100, and 1000 mg/kg bw via gavage
Sampling times: exsanguination via cardiac puncture at 2, 4, 6, 8, 10 and 12 h after administration
Preparation of plasma samples via ultrafiltration using Centricon-10 concentrators
Analysis of plasma samples: HPLC with ion exchange column, detection with in-line radioactivity flow monitor and differential refractive index detector; analyzed metabolites: ethylene glycol, glycolic acid (glycolate), glycolaldehyde, glyoxylic acid (glyoxylate), oxalic acid (oxalate); glyoxalate and glyoxal are not distinguishable in this HPLC system
Limit of quantitation: 33 ng/100 µl of injected plasma filtrate

Incorporation of 14C-actitivity in plasma proteins
Test groups: additional 3 animals/dose level
Concentration: 10, 100, and 1000 mg/kg bw via gavage
Sampling times: serial sampling of 200-300 µl blood via tail vein at 6, 12, 24, 48, and 72 hours post-dosing
Analysis of incorporation of 14C into C1 pool: 15-25 µl of blood samples analysed by liquid scintillation spectrometry (LSS).
Determination of 14C in precipitated protein: remaining blood samples were precipitated with trichloroacetic acid. After centrifugation 14C was determined in the remaining pellet via combustion and counted by LSS.

Profiles of ethylene glycol metabolites in urine
Selected urine samples from 4 rats/dose were analysed for metabolites: selection criterium was the appearance of 14C-activity in total urine.
Analyzed samples, application mode and dose groups:
Intravenous: 10, 1000 mg/kg bw
Peroral (via gavage): 10, 1000 mg/kg bw
Percutaneous: undiluted 10, 1000 mg/kg bw; 50% aqueous solution 1000 mg/kg bw
Sampling times: 0-12 h and 12-24 h intervals with exception of the following application mode: percutaneous absorption, 50% aqueous solution; sampling interval 24-36 hours
Analysis of urine samples: HPLC with ion exchange column, detection with in-line radioactivity flow monitor and differential refractive index detector
Analyzed metabolites: ethylene glycol, glycolic acid (glycolate), glycolaldehyde, glyoxylic acid (glyoxylate), glyoxal, oxalic acid (oxalate)
Limit of quantitation: ca. 1 µg ethylene glycole/ml urine; ca. 2 µg metbolite/ml urine

Pharmacokinetic evaluations
Pharmacokinetic data analysis was based on semilogarithmic plots for concentrations of both total radioactivity and unchanged ethylene glycol in plasma versus time.
Analysis of elimination and transfer of 14C- ethylene glycol between compartments: mean plasma 14C concentrations were used initially to fit one-, two- or three-exponential equations. Both were assumed to be first-order processes.
Plasma concentration-time data:
14C-concentrations after intravenous and dermal application, and ethylene glycol concentration after oral administration: described by a biexponential equation of the form: Ct = AeExp-alpha t + Be Exp-beta t.
14C-concentrations after oral administration described by a triexponential equation and ethylene glycol concentration after intravenous administration described by a monoexponential equation.
Parameters estimated based on mean plasma concentration values:
ka: rate constant for absorption
alpha: rate constant for initial disposition
beta: rate constant for terminal disposition
AUC: area under the curve
Vdss: apparent volume of distribution at steady state
Cl totalEG: total clearance of ethylene glycol;
Cl total = [dose : AUC] x 1000 µg/mg
MRT: mean residence time
U EG: total amount of ethylene glycol excreted in urine from 0-24 h
Cl renal = Cl total x U EG
t½abs: half-time of absorption
t½alpha and t½beta: half-times of elimination
F: fraction of the parent dose absorbed, or its bioavailable dose



Statistics:
means and standart deviation
Type:
other: see detailed text below
Metabolites identified:
yes
Details on metabolites:
see detailed text below

Absorption

Oral route (10, 1000 mg/kg bw):

Plasma concentrations of unchanged ethylene glycol: extremely rapid absorption phase following gavage with half times of absorption t1/2abs of 20-27 (0.3-0.4 h) min at doses of 10 and 1000 mg/kg bw. Absorption followed two-exponential fit of data. Absorption rates were approximately an order of magnitude higher than the elimination rates.

Plasma concentrations of 14C-activity: different kinetics for the two doses. 10 mg/kg bw: Absorption of 14C was moderately rapid with peak plasma concentrations occurring at 4-5 h post dosing. Half time of absorption t1/2abs accounted to 2.7 h. 1000 mg/kg bw: Plasma uptake of 14C was extremely rapid. Peak plasma concentrations were achieved in about 1.2 h post dosing. A half time of absorption t1/2abs of 0.2 h was calculated.

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol):

Plasma concentrations of unchanged ethylene glycol: only quantifiable for the high dose. Time course of absorption was described by a biexponential equation. Peak plasma concentrations were achieved in 11.6 h post-dosing. Half time of absorption t1/2abs accounted to 3.9 h and thus was distinctly higher than after oral administration.

Plasma concentrations of 14C-activity: 1000 mg/kg bw: Time course of absorption was described by a biexponential equation. Peak plasma concentrations were achieved in about 24.4 h. A half time of absorption t1/2abs of 6.7 h was calculated. Plasma curves were similar in the absorption phase between ethylene glycol and total radioactivity, suggesting that ethylene glycol is probably being absorbed through rat skin in the unchanged form. 10 mg/kg bw: Time course of absorption was similar to the high dose. Peak plasma concentrations were achieved about 23 h post-dosing, with a corresponding half time of absorption t1/2abs of 6.1 h.

Peak plasma concentrations of 14C were proportional to dose: 0.4 and 50 µg equ./g.

Total absorption or skin penetration of 14C, respectively, accounted to 31.5% for the low dose and to 35.7% for the high dose.

Dermal route (1000 mg/kg bw, 50 % aqueous dilution of ethylene glycol):

Plasma concentrations of unchanged ethylene glycol: no unchanged ethylene glycol quantifiable over the 96-hour post-dosing period, so that virtually no unchanged ethylene glycol penetrated the skin.

Plasma concentrations of 14C-activity: only about 1% of applied 14C penetrated the skin during the 6-hour contact period. Almost all of the applied dose (> 90%) was recovered as unabsorbed radioactivity.

Total absorption or skin penetration of 14C, respectively, accounted to 22.1%.

Bioavailability of unchanged ethylene glycol

Oral route (10, 1000 mg/kg bw): There was virtually complete bioavailability for unchanged ethylene glycol at both dose levels.

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol; 1000 mg/kg bw, 50 % aqueous dilution of ethylene glycol): Bioavailability (parameter F) for the 1000 mg/kg bw undiluted ethylene glycol dose accounted to 22.5% thus being substantially lower than for oral dosing. In contrast, no bioavailability was demonstrable for the 10 mg/kg bw undiluted dose or the 1000 mg/kg bw 50% aqueous dilution dose.

Elimination from plasma

Intravenous application (10, 1000 mg/kg bw):

Plasma elimination of unchanged ethylene glycol: Disappearance of unmetabolized ethylene glycol from plasma followed monooexponential elimination patterns. Elimination was rapid both at the low and high dose with t1/2beta of 0.8-1.2 h and overall clearances Cl total of 5.3 and 3.0 ml/min/kg, respectively. At the low dose ethylene glycol was detectable in plasma only up to 8 hours post dosing. Maximal concentrations and AUC were roughly dose-proportional. A possible renal tubular reabsorption of ethylene glycol due to bolus i.v. application was discussed based on low values of Vdss, indicating relatively small tissue distributions, and Cl renal. Renal excretion of unchanged ethylene glycol was with values of 0.9-1.2 ml/min/kg lower than the glomerular filtration rate of the rat (3.4 ml/min/kg).

Plasma elimination of 14C-activity: The disappearance of total radioactivity from plasma followed a two-exponential elimination pattern. The initial distribution phase for plasma radioactivity was very brief for the low dose (t1/2beta of 1-2 min) and comparatively rapid for the high dose (t1/2alpha of 3.9 h). This was followed by an extended terminal phase with t1/2beta of 26.6 h and 37.0 h for the low and high dose, respectively. At the 1000 mg/kg bw i.v. dose evidence of non-linear kinetic patterns was suggested from t1/2beta, along with a disproportionately larger AUC versus the low dose.    

Oral route (10, 1000 mg/kg bw):

Plasma elimination of unchanged ethylene glycol: well-described by two-exponential fit of data. Elimination was slower than the rate of absorption, with t1/2betas of 1.4 and 2.0 h for the low and high dose. The last quantifiable time point was at 12 h post-dosing for the low dose. A possible renal tubular reabsorption of ethylene glycol was discussed based on low values of Vdss, indicating relatively small tissue distributions, and Clrenal. Renal excretion of unchanged ethylene glycol was with values of 0.8-0.9 ml/min/kg lower than the glomerular filtration rate of the rat (3.4 ml/min/kg). A linear phamarcokinetic relationship for unchanged ethylene glycol was apparent for both dose levels.

Plasma elimination of 14C-activity: 10 mg/kg bw: Elimination followed a biexponential manner, with a rapid  phase from 6-18 h post-dosing and a slower terminal phase t1/2beta of 39.8 h through 96-h post-dosing. 1000 mg/kg bw: Elimination followed again a biexponential manner, with a rapid alpha phase and a much slower terminal phase t1/2beta of 60.2 h. This resulted in a disproportionately lower AUC than would be expected for the 100-fold increase in dose. A further evaluation of data from dosages of 10, 400, 600, 800, and 1000 mg/kg bw showed that elimination of radioactivity from plasma was virtually comparable for all 5 dosages with half-lifes ranging from 28 to 31 h. Also the values of AUC were roughly proportional to the administered doses.

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol):

Elimination of 14C-activity from plasma showed a similar time course for both doses with values of t1/2beta of 50.4 and 65.9 h for the low and high dose, respectively. Time course of elimination was described by a biexponential equation. Elimination occurred much slower than absorption.

Incorporation of 14C-activity in plasma proteins

Oral route (10, 1000 kg/kg bw): Sustained levels of 14C were found in the plasma protein pellets, whereas a decrease in remaining plasma supernatants was found. Thus, at 24 h 14C concentrations in the pellets were 5-10 fold larger than in the supernatants.

Disposition of 14C-activity in tissues and excreta

Recoveries of total radioactivity were within 81 to 93% with exception of the 10 mg/kg bw dermal application with a reduced recovery of 49% only.

Tissue distribution of 14C-activity

Intravenous and oral route (10, 1000 mg/kg bw):

Total tissue recovery of 14C accounted to 8.5 and 10.1% at the low dose. At the high dose there was a twofold decrease to 4.7-5.0%. Concentrations of 14C in individual tissues showed a dose-dependence being about two orders of magnitude lower at the low dose compared to high dose tissue values. Tissue/plasma ratios > 1.0 were calculated for liver, kidney, lung and pelt, indicating a greater presence of 14C in these tissues than would be accounted by plasma perfusion alone.

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol):

The distribution of 14C to tissues showed similar disposition profiles to those observed after i.v. and p.o. dosing. Tissue/plasma ratios > 1.0 were calculated for: liver, kidney, and pelt at 10 mg/kg bw, for liver, kidney, lung and pelt at 1000 mg/kg bw (undiluted), and for liver and pelt at 1000 mg/kg bw (aqueous dilution), indicating a greater presence of 14C in these tissues than would be accounted by plasma perfusion alone.

Metabolism

Intravenous application (10, 100, 1000 mg/kg bw): At 12 h post-dosing plasma concentrations of unchanged ethylene glycol and the main metabolite glycolate were consistent with concentrations found after oral dosing (see below).

Oral route (10, 100, 1000 mg/kg bw); Metabolites identified in plasma: Along with unchanged ethylene glycol, glycolate constituted the largest percentages of radioactivity for all three dosages detected in plasma over the first 12 hours following dosing. Glycolate was the main metabolite. Besides, at 10 and 100 mg/kg bw glyoxylate/glyoxal and traces of glycoaldehyde were identified. Glycolate and glyoxylate/glyoxal declined steadily over the first 12 hours post-dosing. At 1000 mg/kg bw, besides glycolate only glyoxylate/glyoxal was found and only at the first time point 4 h after dosing. The glycolate concentration remained at an almost constant level at the high dose.

Time course of metabolite profiles in plasma (up to 12 h post-dosing)

unmetabolized ethylene glycol: t1/2beta 1.37, 1.29, and 2.01 h at 10, 100, and 1000 mg/kg bw, respectively

Glycolate: 10 and 100 mg/kg bw: relative rapid decrease with t1/2beta of 3.7 h and 0.95 h, respectively; 1000 mg/kg bw: relative constant level due to t1/2beta of 17.6 h

Glyoxylate/glyoxal: 10 and 100 mg/kg bw: steady decline; t1/2beta was 2.45 h at 10 mg/kg bw; 1000 mg/kg bw: only detectable at 4 h

Dermal route (1000 mg/kg bw, undiluted ethylene glycol): There are indications that some of the applied ethylene glycol dose is metabolised prior to excretion in urine as Cmax and tmax for the unchanged ethylene glycol plasma curve showed a faster time course than for 14C-activity (30.4 µg/g vs. 50.0 µg/g, and 11.6 h vs. 24.4 h; values of unchanged ethylene glycol vs. 14C-activity), and the AUC showed substantial differences (1249 µg/g h vs. 6127 µg equ./g h).

Excretion

Total excretion of radioactivity

14C-activity was recovered from urine, faeces and as 14CO2 in exhaled air in percentages varying for different application routes and doses.

Intravenous and oral route (10, 1000 mg/kg bw): 10 mg/kg bw: major excretion route: exhalation 41-42%; urine: 26-27%; faeces: 2.9%; 1000 mg/kg bw (values for i.v. and p.o. route): major excretion route: urine 52.5%, 42.7%; exhalation 28.2%, 27.3%; faeces: 2.8%, 4.4%

Oral route (400, 600, 800 mg/kg bw): In the dose range of 400 to 800 mg/kg bw excretion via exhalation of 14CO2 was the main route. There was a dose-dependent increase of urinary excretion being accompanied by a decrease of 14CO2 exhalation.

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol): 10 mg/kg bw: major excretion route: exhalation 14.0%; urine 6.7%; faeces 1.1%. 1000 mg/kg bw: exhalation 14.4%; urine 8.1%; faeces 0.6%. Dermal route (1000 mg/kg bw, undiluted ethylene glycol): major excretion route: exhalation 5.9%; urine 4.6%; faeces 0.6%.

Time course of excretion of radioactivity and metabolites

Intravenous and oral route (10, 1000 mg/kg bw):

Elimination profiles for total radioactivity were similar in both time-course and amount of the dose excreted for both application routes and for each dosage, indicating that the majority of the oral doses were absorbed. For both routes, at the low dose excretion of radioactivity was mainly via exhalation as 14CO2. In contrast, at the high dose urinary excretion was the dominant route. Urinary excretion: majority of 14C excreted within 12 h, urinary excretion virtually complete within 24-36 h post-dosing. At the high 1000 mg/kg bw-dose a higher percentage was excreted via urine (i.v. 54%, p.o. 43%) than after the low 10 mg/kg bw-dose (27%). 10 mg/kg bw: Urinanalysis of metabolites yielded a similar pattern for the low dose for both application routes with unchanged ethylene glycol accounting to 89-96% of total 14C. During the first 12 h glycolic acid and oxalic acid were found as minor metabolites (ca. 7% in total) whereas during the 12-24 h interval 11% (i.v.) and 4% (p.o.) accounted to an unidentified metabolite. 1000 mg/kg bw: During the first 12 h glycolic acid excretion increased to 35% (i.v.) and 25% (p.o.) besides unchanged ethylene glycol. During the 12-24 h interval excretion of unchanged ethylene glycol decreased to 38% (i.v.) and 55% (p.o.) whereas oxalic acid appeared as second metabolite (17% and 7%) besides glycolic acid (45% and 38%).

Exhalation of 14CO2: There was still a slow increase of cumulative exhaled 14CO2 at the end of the 96-h observation period. Within this interval the majority of 14CO2 was exhaled within the first 24 h. Overall levels were inversely related to dose (43% at the low dose, 28-30% at the high dose).

Dermal route (10, 1000 mg/kg bw, undiluted ethylene glycol): Elimination profiles for total radioactivity showed a continuous increase of the amount of the dose excreted during the 96-h observation period for each dosage. Excretion via exhalation was higher than via urine with comparable amounts excreted for both doses. Total excretion was lower than after i.v. or p.o. application. However, taking the low dermal penetration into account, excreted amounts corresponded to 25-35% of the absorbed doses and thus were comparable with excretion percentages after i.v. or p.o. dosing. Urinary excretion: increasing continuously during 96-h observation period up to a final amount of 8-9% of the dose. During the first 12 h 14C was completely excreted as glycolic acid, during the 12-24 h interval glycolic acid excretion decreased to ca. 3 and 13% (low and high dose) of total 14C, whereas 97% and 87% accounted to unchanged ethylene glycol.

Exhalation of 14CO2: increasing continuously during 96-h observation period up to a final amount of 15-17% of the dose

Dermal route (1000 mg/kg bw, 50 % aqueous dilution of ethylene glycol): Measurable excretion of 14C was retarded until 24 h post-dosing. There was a continuous increase during the 96-h observation period until final levels of ca. 5% of the applied dose for each of the excretion routes. The 24-36-hour interval was the first interval in which radioactivity was quantifiable in urine. This accounted to 100% to unchanged ethylene glycol.

Conclusions:
No bioaccumulation potential based on study results
Ethylene glycol given by three different routes demonstrated apparent first-order toxicokinetic behaviour for disposition in and the elimination from plasma.
Executive summary:

The applied test method was comparable to OECD guideline 417. 14C-ethylene glycol was administered to male Sprague-Dawley rats (n=6 per group) intravenously, orally or dermally in single doses ranging from 10 – 1000 mg/kg bw. Toxicokinetic investigations of the fate of ethylene glycol included absorption from the gastrointestinal tract, skin penetration, plasma clearance, tissue distribution, identification of metabolites in plasma and urine, and pathways and kinetics of excretion.

Depending on the route of administration distinct differences in the amount of absorbed 14C ethylene glycol are found.

After oral application ethylene glycol is absorbed very rapidly and almost completely from the gastrointestinal tract based on the short half time of 0.3-0.4 h and virtually complete bioavailability of unchanged ethylene glycol. Similar 14C concentrations in tissues after p.o. and i.v. application give additional evidence of virtually complete absorption.

Absorption of ethylene glycol via rat skin is slower with half-times of ca. 4 h. Total absorbed radioactivity accounted to only ca. 31-36 % after dermal application of undiluted ethylene glycol, and to 22.1 % when applying the 50 % aqueous solution. Bioavailability of unchanged ethylene glycol was 22% for 1000 mg/kg bw (undiluted ethylene glycol).

Relative recoveries of 14C in tissues and carcass were higher at the 10 mg/kg bw p.o. and i.v. dose in comparison to the 1000 mg/kg bw dose, whereas individual 14C concentrations in tissues increased dose-dependently. Storage in tissue accounted to total amounts of ca. 4-10% of the dose after 96 hours.

Along with unchanged ethylene glycol, glycolate constituted the largest amount of radioactivity in plasma up to doses of 1000 mg/kg bw during the first 12 hours following dosing. Besides, glyoxylate/glyoxal was identified as metabolite and, up to doses of 100 mg/kg bw, traces of glycoaldehyde. Clearance of unchanged ethylene glycol followed first-order kinetics with half times ranging from 1.29-2.09 h for all doses. Glycolate clearance was rapid at low doses (plasma half time 0.95 and 3.7 h) and distinctly retarded at 1000 mg/kg bw (half time 17.6 h). 

During 96 h after oral or i.v. application, at the low dose of 10 mg/kg bw, ethylene glycol is extensively metabolised via oxidative pathways and exhaled as CO2 (about 42 % of the dose). Expiration of 14CO2 diminishes to ca. 28 % as the dose increases to 1000 mg/kg bw. 26-53 % of the dose are excreted in the urine. Glycolate was the major urinary metabolite besides minor amounts of oxalate. Amounts of both metabolites were increased in urine with increasing dose indicating saturation of the metabolism of glycolate to glyoxylate. Only minor amounts of 14C-activity are both excreted in the faeces (2.4-2.9 %) and remain in the carcass (5-11.6 %). Conclusion: Ethylene glycol given by three different routes demonstrated apparent first-order toxicokinetic behaviour for disposition in and the elimination from plasma.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Objective of study:
metabolism
GLP compliance:
not specified
Species:
other: data from different mammalian species including human data
Details on dosing and sampling:
.

Ethylene glycol is converted to glycolaldehyde by nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase. Subsequent reduction of NAD results in the formation of lactic acid from pyruvate. Glycolaldehyde has a brief half-life and is rapidly converted to glycolic acid (and to a lesser extent glyoxal) by aldehyde dehydrogenase and aldehyde oxidase, respectively. Glycolic acid is oxidized to glyoxylic acid by glycolic acid oxidase or lactic dehydrogenase. Glyoxylic acid can be metabolized to formate, glycine, or malate, all of which may be further broken down to generate respiratory CO2, or to oxalic acid, which is excreted in the urine. In excess, oxalic acid can form calcium oxalate crystals. Rate-limiting steps in the metabolism of ethylene glycol include the initial formation of glycolaldehyde and the conversion of glycolic acid to glyoxylic acid, both of which are saturable processes. The conversion of glycolic acid to glyoxylic acid is the most rate-limiting step in ethylene glycol metabolism. Both glycolic acid and oxalic acid are found in the blood and urine of unexposed individuals as a result of normal metabolism of proteins and carbohydrates (plasma glycolic acid 0.0044–0.0329 mM and in urine 0.075–0.790 mM).

Conclusions:
Ethylene glycol is initially metabolized to glycolaldehyde by alcohol dehydrogenase; glycolaldehyde is rapidly converted to glycolate and glyoxal by aldehyde oxidase and aldehyde dehydrogenase; metabolism of glycolate by glycolate oxidase or lactate dehydrogenase results in the formation of glyoxylate, which may be further metabolised to formate, oxalate, glycine, and CO2.

Description of key information

The test substance hydrolyses to ethylene glycol and formaldehyde.

In rats, ethylene glycol is very rapidly and almost completely absorbed after oral doses but dermally-applied EG was slowly and rather poorly absorbed; oxidative metabolite pathways appeared to be saturated at high oral doses; linear pharmacokinetic relationship was apparent for the oral route. In mice, ethylene glycol is completely and rapidly absorbed after oral application, absorption via skin is lower and accounted to ca. 40-50% of applied dose.

Ethylene glycol is initially metabolized to glycolaldehyde by alcohol dehydrogenase; glycolaldehyde is rapidly converted to glycolate and glyoxal by aldehyde oxidase and aldehyde dehydrogenase; metabolism of glycolate by glycolate oxidase or lactate dehydrogenase results in the formation of glyoxylate, which may be further metabolised to formate, oxalate, glycine, and CO2.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Data on formaldehyde

Formaldehyde is an essential metabolic intermediate in humans as well as in animals (IARC 1995). After inhalation formaldehyde is absorbed and deposited in the upper respiratory tract, the site of first contact. The localisation of uptake in each species is determined by nasal anatomy, mucus coating and clearance mechanisms (WHO 2002). The overall uptake by the nasal passages at resting airflow rates has been predicted to be 90% in rats, 67% in monkeys, and 76% in humans (BfR 2006). The physiological level of formaldehyde in the blood of humans and experimental animals is not increased after inhalation exposure due to its rapid oxidation to formic acid and reactivity at the site of first entry (Heck et al. 1985, Casanova et al. 1988).

Formaldehyde is rapidly and nearly completely absorbed from the intestinal tract after oral exposure (Buss et al. 1964). After dermal application to rats and guinea pigs ca. 30-40% of the applied formaldehyde is absorbed via the skin; the skin of monkeys is less permeable (Jeffcoat et al. 1983). Further experiments in rats resulted in absorptions rate below 10% of the applied dose (Bartnik et al. 1985). However, a concentration of 0.1% formaldehyde in a cream was applied and methodological shortcomings are not excluded.

The data available on metabolism and biological pathways of formaldehyde are summarized according to BfR (2006). Formaldehyde reacts spontaneously and non-enzymatically with glutathione to form S-hydroxymethylglutathione (1). In the presence of NAD+, S-hydroxymethyl­glutathione can be converted to formylglutathione (2) catalysed by formaldehyde dehydrogenase (FAD). In the presence of water, formylglutathione can be cleaved by S-formylglutathione hydrolase to glutathione and formic acid (3). Formic acid can be excreted as its sodium salt via urine or cleaved to CO2and exhaled. As formate an uptake into the carbon-1-metabolic pathway is also possible [see also (9)].

Other biological pathways are: Formaldehyde can be reversibly bound to cysteine to form thiazolidine-4-carboxylate (4). Formaldehyde reacts reversibly with urea or protein to form hydroxymethyl-adducts (5) or protein-adducts (6), respectively. Irreversible reaction with two proteins results in protein-protein-cross-links (7) and with DNA and protein in DNA-protein-cross-links (8). The uptake into the one-carbon metabolic pathway is possible after non-enzymatical binding of formaldehyde to tetrahydrofolic acid (9). In this activated form formaldehyde is an essential intermediate for the synthesis of purine, thymidine and certain amino acids which are incorporated in proteins and DNA.

The glutathione dependent oxidation of formaldehyde to formic acid catalysed by formaldehyde dehydrogenase is considered to be the main defence mechanism against the formation of covalent binding of formaldehyde to macromolecules like proteins or DNA. After depletion of glutathione also other oxidation pathways, catalysed by aldehyde dehydrogenase and catalase, may become important (Casanova-Schmitz et al. 1984, BfR 2006). The metabolite formic acid can be excreted as its sodium salt via urine or cleaved to CO2and exhaled. An uptake into the carbon-1-metabolic pathway is also possible (BfR 2006). In inhalation studies in rats using14C-labelled formaldehyde 40% of applied radioactivity was excreted within the following 70 hours via exhalation, 17% via urine and 5% via faeces (Heck et al. 1983).