Reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: basic information given.

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Objective of study:

excretion

Principles of method if other than guideline:

Mortality and urinary excretion of oxalate.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: gavage

Duration and frequency of treatment / exposure:

The daily average urinary excretion of oxalate was measured on 70 control rats after 24 hours of fasting.

Remarks:

Doses / Concentrations:
Each trial group was given a single LD50 dose of the test substance (15 ml/kg of body weight).

No. of animals per sex per dose / concentration:

Groups 1, 2, 3, 4, 5: 30 rats; groups 6 and 7: 15 rats

Details on dosing and sampling:

The urinary concentration of oxalate was determined by the colorimetric method of Hodgkinson and Williams. The analysis of the calcium oxalate crystals was made on the sediment of the daily urine output with a polarizing microscope and the birefractive crystals were counted by fields (ohne approx. for the high score).
First, the daily average urinary excretion of oxalate was measured on 70 control rats after 24 hours of fasting. Then, each trial group was given a single LD50 dose of the test substance (15 ml/kg of body weight as according to Fitzhugh and Nelson) by a gastroesophageal tube. Urinary excretion of oxalate was then determined for two days and mortality was noted each day until the animals were sacrificed for histological studies 5 days after the intoxication. Seven groups were tested; for details, see table below.

Statistics:

The statistical analysis was performed using the Student's t test for small samples.

Toxicokinetic parameters:

other: Acute intoxication by diethylene glycol (LD 50) in male rats is associated with a considerable urinary excretion of oxalate, which is significantly decreased by alkalinisation and/or intraperitoneal injection of ethanol with hydration.

Metabolites identified:

not measured

Details on metabolites:

no data

Bioaccessibility (or Bioavailability) testing results:

no data

Conclusions:

Acute intoxication by diethylene glycol (LD 50) in male rats is associated with a considerable urinary excretion of oxalate, which is significantly decreased by alkalinisation and/or intraperitoneal injection of ethanol with hydration. Mortality during the five days following intoxication is significantly decreased by major hydration only or together with pyridoxine administration, but is cancelled by major hydration together with alkalinisation or intraperitoneal administration of ethanol, plus hydration, with or without alkalinisation. It might be inferred that diethylene glycol has the same metabolic pathway as ethylene glycol and treatment of acute intoxication by diethylene glycol should be the same as that of acute poisoning with ethylene glycol.

Reference 2

Endpoint:

dermal absorption in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study conducted was not in accordance to any guideline/s and GLP, there is sufficient data to permit meaningful evaluation of the study results.

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Qualifier:

no guideline available

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Type of coverage:

not specified

Vehicle:

other: methanol

Duration of exposure:

15 minutes, 64 minutes, 8 hours and 24 hours

No. of animals per group:

4 rats received 75 µl of diethylene glycol in methanol

Conclusions:

Cryostat sectioning showed that diethylene glycol is absorbed through the skin of rats based on percent of radioactivity remaining in the dosed area, which was 47.2, 32.6, 29.2 and 16.8 per cent after contact times of 15 minutes, 64 minutes, 8 hours and 24 hours, respectively. After 15 minutes post dermal administration of diethylene glycol, a radioactive peak appeared ~ 112 microns into the skin. Percent of radioactivity in the urine after contact times of 8 and 24 hours was 11.9 and 32.2 per cent, respectively

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication, meets generally accepted scientifically principles

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Objective of study:

other: metabolism and toxicity

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were treated by oral gavage with water, 2 g/kg bw DEG (low dose), 10 g/kg bw DEG (high dose), or 10 g/kg bw DEG + fomepizole, and blood and urine were collected over 48 h to investigate the mechanism for the acute toxicity of DEG, and the effect of the alcohol dehydrogenase inhibitor 4-methylpyrazole (fomepizole), by determining the relationship between accumulation of DEG or its metabolites and the resulting kidney and liver toxicity.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, indianapolis, IN
- Age at study initiation: adult animals were used
- Weight at study initiation: 425–475 g
- Housing: in metabolic cages for 48 h for urine collection
- Individual metabolism cages: yes
- Diet: ad libitum: yes
- Water: ad libitum: yes


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

no data

Duration and frequency of treatment / exposure:

- DEG was administered once by oral gavage at time 0
- Fomepizole (15 mg/kg bw) was administered ip as a 10 mg/ml solution in saline at 15 min and then at 12, 24, and 36 h (10 mg/kg bw) for a total of four ip doses

Remarks:

Doses / Concentrations:
- 2 g/kg bw or 10 g/kg bw DEG
- 10 g/kg bw DEG + Fomepizole (15 mg/kg bw)

No. of animals per sex per dose / concentration:

Six male rats per group

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

The purpose of this study was to investigate the mechanism for the acute toxicity of DEG, and the effect of the alcohol dehydrogenase inhibitor 4-methylpyrazole (fomepizole), by determining the relationship between accumulation of DEG or its metabolites and the resulting kidney and liver toxicity.

Details on dosing and sampling:

- Urine was collected at 4, 8, 12, 24, 36, and 48 h over ice to minimize degradation of urinary metabolites
- Approximately 1 mL of blood was collected via the indwelling jugular catheter at 4, 8, 12, 24, 36, and 48 h into heparinized syringes

Statistics:

Differences between treatment groups and time points were assessed with two-way ANOVA with Bonferroni post-hoc test. To compare differences between treatment groups only, one-way ANOVA with Tukey post-hoc test was used. All analyses were performed using GraphPad Prism 5 for Windows. Tests were considered significant if p < 0.05.

Preliminary studies:

Not applicable

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Metabolites identified:

yes

Details on metabolites:

After low and high doses of DEG, 2-hydroxyethoxyacetic acid (HEAA) was the primary metabolite in the urine, with only minor amounts of urinary diglycolic acid (DGA). Small amounts of ethylene glycol (EG), but not oxalate or glycolate, were observed in the urine.

Bioaccessibility (or Bioavailability) testing results:

Not applicable

Rats treated with highdose DEG had metabolic acidosis, increased blood urea nitrogen and creatinine, and marked kidney necrosis, noted by histopathology. A minor degree of liver damage was noted at the high dose.

Treatment with fomepizole blocked the formation of HEAA and DGA and the development of metabolic acidosis and the kidney and liver toxicity.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication, meets generally accepted scientifically principles

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Objective of study:

other: metabolism and toxicity

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of this study was to investigate the accumulation of specific metabolites in blood and target organ tissues and to determine the relationship between tissue accumulation of metabolites and the resulting toxicity. Wistar rats were treated with water, 2 g/kg bw DEG (low dose), 10 g/kg bw DEG (high dose), or 10 g/kg bw DEG + fomepizole (to inhibit DEG metabolism), and blood and tissue samples were collected up to 48 h.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, indianapolis, IN
- Age at study initiation: adult animals were used
- Weight at study initiation: 425–475 g
- Housing: in metabolic cages for 48 h for urine collection (after 8 - 10 h acclimation)
- Diet: ad libitum: yes
- Water: ad libitum: yes


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Photoperiod (hrs dark / hrs light): 12/12
- Humidity (%): 50 ± 10

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

no data

Duration and frequency of treatment / exposure:

- DEG was administered once by oral gavage at time 0
- Fomepizole (15 mg/kg bw) was administered ip as a 10 mg/ml solution in saline at 15 min and then at 12, 24, and 36 h (10 mg/kg bw) for a total of four ip doses

Remarks:

Doses / Concentrations:
- 2 g/kg bw or 10 g/kg bw DEG
- 10 g/kg bw DEG + Fomepizole (15 and 10 mg/kg bw)

No. of animals per sex per dose / concentration:

Six male rats per group

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

The purpose of this study was to investigate the accumulation of specific metabolites in blood and target organ tissues and to determine the relationship between tissue accumulation of metabolites and the resulting toxicity. Wistar rats were treated with water, 2 g/kg bw DEG (low dose), 10 g/kg bw DEG (high dose), or 10 g/kg bw DEG + fomepizole (to inhibit DEG metabolism), and blood and tissue samples were collected up to 48 h.

Details on dosing and sampling:

- Urine was collected at 4, 8, 12, 24, 36, and 48 h over ice to minimize degradation of urinary metabolites.
- Approximately 1 mL of blood was collected via the indwelling jugular catheter at 4, 8, 12, 24, 36, and 48 h into heparinized syringes.

Statistics:

Values in the text represent the group mean value ± SEM.
Differences between treatment groups and time points were assessed with twoway ANOVA with Bonferroni post hoc test. To compare differences between treatment groups only, one-way ANOVA with Tukey post hoc test was used. All analyses were performed using GraphPad Prism 5 for Windows. Tests were considered significant if p < 0.05.

Preliminary studies:

Not applicable

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Metabolites identified:

yes

Details on metabolites:

After high doses of DEG, 2-hydroxyethoxyacetic acid (HEAA) was the primary metabolite in the blood (ca. 4 mmol/L), with only low concentrations of diglycolic acid (DGA) - ca.0.04 mmol/L.
In contrast, renal and hepatic concentrations of DGA and of HEAA at 48 h were similar (ca.4 mmol/L), indicating a 100-fold concentrative uptake of DGA by kidney tissue.

Treatment with fomepizole blocked the formation of HEAA and DGA and the kidney toxicity. Both HEAA and DGA concentrations in the kidney correlated strongly with the degree of kidney damage. Accumulation of HEAA in blood correlated with increased anion gap and decreased blood bicarbonate so appeared responsible for the DEG-induced acidosis.

Reference 5

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication, meets generally accepted scientifically principles

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Objective of study:

other: metabolism + toxicity

Qualifier:

no guideline followed

Principles of method if other than guideline:

Human proximal tubule (HPT) cells in culture, obtained from normal cortical tissue were incubated with increasing concentrations of DEG, 2-HEAA, or DGA separately and in combination for 48 h at pH 6 or 7.4, and various parameters of necrotic and apoptotic cell death were measured.

GLP compliance:

no

Radiolabelling:

no

Species:

other: not applicable, in vitro test

Strain:

other: not applicable, in vitro test

Sex:

not specified

Details on test animals or test system and environmental conditions:

not applicable, in vitro test

Route of administration:

other: not applicable, in vitro test

Vehicle:

other: growth medium

Details on exposure:

Growth media was removed, and confluent HPT cell cultures were washed twice with warm PBS. Each chemical (DEG, 2-HEAA, or DGA (all at 0–100 mmol/L)) was first dissolved in growth media, which were then adjusted to pH 6 or 7.4 by the addition of HCl or NaOH (to mimic acidified or neutral conditions); these media were added to separate wells in triplicate, and the cells were incubated for up to 48 h under standard conditions (5 % CO2, 90 % humidity, 37 °C).

Duration and frequency of treatment / exposure:

Cells were incubated for up to 48 h.

Remarks:

Doses / Concentrations:
Cells were incubated at a concentration range of 0 - 100 mmol/L for each test item

No. of animals per sex per dose / concentration:

Not applicable

Control animals:

other: not applicable

Positive control reference chemical:

no

Details on study design:

Human proximal tubule (HPT) cells in culture, obtained from normal cortical tissue were incubated with increasing concentrations of DEG, 2-HEAA,
or DGA separately and in combination for 48 h at pH 6 or 7.4, and various parameters of necrotic and apoptotic cell death were measured:
- Cell exposures to assess necrotic and apoptotic cell death
- Measurement of ethidium homodimer (EtHD) uptake
- Measurement of LDH release
- Measurement of caspase-3 activity
- Cell death detection ELISA
- Measurement of annexin V and propidium iodide staining
- Measurement of ATP levels
- Measurement of succinate dehydrogenase activity
- Cell exposures with potential DGA uptake inhibitors

Statistics:

Data were compared by one-way ANOVA, with either Tukey’s or Dunnett’s post hoc tests to compare differences between individual groups or differences between individual groups and the control, respectively.
A significance level of a <0.05 was used. All analyses were performed using GraphPad Prism 5 for Windows. Single experiments were conducted using triplicate wells of a single cell isolate per concentration of compound. Experiments were then replicated with multiple cell isolates (tissue source).
Results are expressed as a mean of multiple experiments ± SEM, where n represents the number of tissue sources.

Preliminary studies:

Not applicable

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Details on metabolites:

Not applicable

Bioaccessibility (or Bioavailability) testing results:

Not applicable

Reference 6

Endpoint:

basic toxicokinetics, other

Remarks:

MAK documentation, 1995

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Specific details on test material used for the study:

Diethylene glycol, CAS 111-46-6

Details on absorption:

After oral uptake DEG is rapidly absorbed by rats and distributed in all tissues (Heilmair et al. 1993). About 10% of the dermally applied dose is absorbed in rats (Mathews et al. 1991).

Details on excretion:

The half life of DEG in the blood is about 3.5 hours; 73-96% of a radiolabelled oral dose was excreted in the urine.

Details on metabolites:

After a single high dose administration of DEG, no metabolism to monoethylene glycol or oxalate occurs in rats (Heilmair et al. 1993; Lenk et al. 1989; Matthews et al. 1991; Wiener and Richardson 1989). Increased oxalate excretion in the urine of male rats has been observed in long term experiments (Gaunt et al. 1976). The main metabolite is 2-hydroxyethoxyacetic acid.

Reference 7

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication, meets generally accepted scientifically principles

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Qualifier:

no guideline followed

Principles of method if other than guideline:

DEG and its metabolites in stored serum, urine, and cerebrospinal fluid (CSF) specimens obtained from human DEG poisoning victims enrolled in a 2006 case control study were quantitatively measured and analysed. Analytes were measured using low resolution GC/MS, descriptive statistics calculated and case results compared with controls when appropriate. Specimens were deidentified so previously collected demographic, exposure, and health data were not available.

GLP compliance:

no

Radiolabelling:

no

Species:

human

Strain:

other: not applicable

Sex:

not specified

Details on test animals or test system and environmental conditions:

Not applicable

Details on exposure:

Not applicable.
Biological samples (serum, urine, and CSF) used in this study were obtained during an epidemiological investigation of a DEG mass poisoning in the Republic of Panama in 2006.

Duration and frequency of treatment / exposure:

Not applicable

Remarks:

Doses / Concentrations:
Not applicable

No. of animals per sex per dose / concentration:

Not applicable

Positive control reference chemical:

Not applicable

Details on study design:

Not applicable

Details on dosing and sampling:

Not applicable

Preliminary studies:

Not applicable

Details on metabolites:

Diglycolic acid is associated with human DEG poisoning and may be a biomarker for poisoning.

The following samples were analyzed: serum, 20 case, and 20 controls; urine, 11 case and 22 controls; and CSF, 11 samples from 10 cases and no controls.

Diglycolic acid was detected in all case serum samples (median, 40.7 mcg/mL; range, 22.6 – 75.2) and no controls, and in all case urine samples (median, 28.7 mcg/mL; range, 14 – 118.4) and only five (23%) controls (median, < Lower Limit of Quantitation (LLQ); range, LLQ – 43.3 mcg/mL). Significant differences and associations were identified between case status and the following: 1) serum oxalic acid and serum HEAA (both OR < 14.6; 95% CI = 2.8 – 100.9); 2) serum diglycolic acid and urine diglycolic acid (both OR>999; exact p < 0.0001); and 3) urinary glycolic acid (OR = 0.057; 95 % CI = 0.001 – 0.55). Two CSF sample results were excluded and two from the same case were averaged, yielding eight samples from eight cases. Diglycolic acid was detected in seven (88 %) of case CSF samples (median, 2.03 mcg/mL; range, < LLQ, 7.47).

Significantly elevated HEAA (serum) and diglycolic acid (serum and urine) concentrations were identified among cases, which is consistent with animal data. Low urinary glycolic acid concentrations in cases may have been due to concurrent AKI. Although serum glycolic concentrations among cases may have initially increased, further metabolism to oxalic acid may have occurred thereby explaining the similar glycolic acid concentrations in cases and controls. The increased serum oxalic acid concentration results in cases versus controls are consistent with this hypothesis.

Conclusion:

Diglycolic acid is associated with human DEG poisoning and may be a biomarker for poisoning.

Reference 8

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

Prior to or equal to 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Unknown whether GLP study. No Guideline available. Sufficient data available for interpretation of results.

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Objective of study:

metabolism

Qualifier:

no guideline available

GLP compliance:

not specified

Radiolabelling:

no

Species:

other: horse

Strain:

not specified

Sex:

not specified

Route of administration:

other: in vitro assay

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

No data.

Remarks:

Doses / Concentrations:
No data.

No. of animals per sex per dose / concentration:

Not applicable.

Control animals:

other: in vitro assay run without alkaline phosphatase and the inhibitor of alkaline phosphatase, 4-methylpyrazole

Preliminary studies:

Not applicable

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Metabolites identified:

no

Details on metabolites:

There was no appreciable reaction in the absence of alcohol dehydrogenase. Reaction was completely inhibited by 4-methylpyrazole. Vmax and Km values are tabulated below.

Bioaccessibility (or Bioavailability) testing results:

Not applicable.

Table 1 Vmax and Km values in in vitro equine alkaline phosphatase studies

Substrate	Km (mM)	Vmax (nmol/min)
EG	1000 ± 60	64 ± 1
DEG	1900 ± 300	27 ± 5
TEG	810 ± 50	19 ± 2
TetraEG	490 ± 20	17 ±1
PentaEG	340 ± 20	12 ± 2

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
The ethylene glycols are in vitro substrates for alcohol dehydrogenase. Vmax for metabolism of the ethylene glycols consistently decreases with the number of glycol units. Km consistently decreased with the number of glycol units with the exception of DEG

Executive summary:

The ability of equine alkaline phosphatase to degrade various ethylene glycol compounds was examined in vitro.

The ethylene glycols are in vitro substrates for alcohol dehydrogenase. Vmax for metabolism of the ethylene glycols consistently decreases with the number of glycol units. Km consistently decreased with the number of glycol units with the exception of DEG

Reference 9

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

No data.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to guideline/s and GLP but the report contains sufficient data for interpretation of study results.

Justification for type of information:

Read across based on category approach. Please see category document for further information.

Objective of study:

other: excretion and metabolism

Qualifier:

no guideline available

Principles of method if other than guideline:

Triethylene glycol was administered by oral gavage to rats and rabbits. Urine, feces and carbon dioxide were collected and analyzed for potential metabolites.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

Randomly labeled triethylene glycol was synthesized by reaction of 14C ethylene glycol with ethylene oxide.

Species:

rabbit

Strain:

New Zealand White

Sex:

female

Details on test animals or test system and environmental conditions:

New Zealand White female rabbits were obtained from J. C. Heath, Ashland, Virginia.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Nonisotopic triethylene glycol was given by stomach tube to New Zealand White female rabbits in 25% W/V aqueous solution.

Duration and frequency of treatment / exposure:

One rabbit received a single gavage dose while the second received 3 daily doses.

Remarks:

Doses / Concentrations:
2.0 g/kg of triethylene glycol

No. of animals per sex per dose / concentration:

2 rabbits/dose level

Control animals:

not specified

Positive control reference chemical:

No data.

Details on study design:

Administration of Triethylene Glycol and Excretion by Animals Nonisotopic triethylene glycol was given by stomach tube to New Zealand White female rabbits in 25% W/V aqueous solution. Urine was collected over sodium fluoride as runoff from metabolism cages, and all animals were allowed food and water ad libitum during the course of the experiment. Urine samples were used directly or stored frozen.

Fractionation of Urine after Administration of Nonisotopic Triethylene Glycol
Following administration of triethylene glycol, urine from rabbits was made alkaline to pH 12-13 by addition of sodium hydroxide. The urine was then exhaustively extracted with chloroform as described in the section on control studies. The chloroform extract contained triethylene glycol which was determined calorimetrically. An additional sample was then subjected to acidic hydrolysis as described in the section on control studies. The urine was then made alkaline as above for extraction of total triethylene glycol (free plus that from hydrolysis of any glucuronide ester) by chloroform. Some urine samples were acidified to pH 2-3 and then extracted with chloroform to obtain material, not extractable from alkaline solution, putative 0- (P-hydroxyethyl)oxyethoxyacetic acid, which was converted into a chromatogen by reaction with 3,.5-dinitrobenzoyl chloride for the calorimetric determination.

Vapor Phase Chromatographic Determinati,on of an Acid Derived from the Metabolism of Triethylene Glycol
The chloroform solution obtained by a continuous exhaustive extraction (72 hours) of acidified rabbit urine obtained after administration of triethylene glycol was evaporated to dryness. The residue was transferred to a 50-ml glass-stoppered bottle with the aid of 2 ml of absolute methanol. After addition of 0.2 ml of concentrated hydrochloric acid and 2,2-dimethoxypropane (cf. Lorette and Brown, 1959), the mixture was shaken and allowed to stand at room temperature for 1 hour. An aliquot (0.2 ml) was chromatographed by the preceding procedure for vapor phase chromatographic determination of triethylene glycol in urine. The recorder response was equivalent to 1% by weight of triethylene glycol in the urine (approximately 3 times that obtained from a similar extraction of alkalinized urine). The aqueous residue from extracted alkalinized sample was then acidified to pH l-2 by addition of hydrochloric acid, The acidic solution was then exhaustively extracted with chloroform. The chloroform solution which contained no triethylene glycol was then evaporated and subjected to the esterification procedure (above). The esterified product had a retention time (8.5 minutes) on the Carbowax column which was identical to that of triethylene glycol. In comparison the dimethyl ester of ethylenedioxydiacetic acid had a retention time of 7.5 minutes. The esterified metabolite, collected from the column, and the dimethyl ester showed a similar infrared absorption spectrum. The former, however, showed a strong OH absorption at 2.9 u.
.
Another chloroform solution from the exhaustive extraction of the acidified urine (5 ml) was evaporated and then subjected to esterification with methanol (see above). To the residue obtained by evaporation of the esterification mixture was added 2 ml of acetic anhydride. The mixture was heated to 90 C for one-half hour. An aliquot (0.1 ml) was subjected to chromatography in the Perkin-Elmer Vapor Fractometer equipped with a column Q17 (2 meters packed with Apiezon L on Chromosorb Red). The column was adjusted to 200 C at a helium flow rate of approximately 150 ml/min. The peak at a retention time of 16 minutes, corresponding to acetylated triethylene glycol, indicated a triethylene glycol content corresponding to that obtained by chromatography of the chloroform extract of alkalinized urine. An earlier peak at 10.5 minutes differed from that obtained of an authentic sample of the dimethylester of ethylenedioxydiacetic acid (13 minutes). The peak responses of the acetylated and esterified metabolic triethylene glycol (0.6870 by weight in the urine) and that of the acetylated unmetabolized triethylene glycol of the sample (0.31% by weight in the urine) were in good agreement with the results obtained above on the Carbowax column. Evidence that triethylene glycol was determined as an acetyl derivative and that the metabolite was determined as an acetyl methyl ester was confirmed in experiments in which it was shown that neither triethylene glycol, ethylenedioxydiacetic acid, nor the methyl ester of the metabolite would pass through column Q, even when the temperature was raised to 220C.

References:
LORETTE, N. B., and BROWN, J. H., JR. (1959). Use of acetone dimethyl acetal in preparation of methyl esters. J. Org. Ckem. 24, 261.262.

Details on dosing and sampling:

Rabbits were dosed with 2 g/kg of non-labelled triethylene glycol

Statistics:

No data.

Details on absorption:

No data.

Details on distribution in tissues:

No data.

Details on excretion:

Following the oral administration of triethylene glycol (200 mg/kg) to the rabbit, 34.3% of the dose was excreted in the urine as unchanged triethylene glycol. Following a 2000 mg/kg oral dosey for 3 consecutive days, an average of 27.3% (calorimetric analysis) of the total dose was excreted as TEG.

Metabolites identified:

yes

Details on metabolites:

After oral doses of triethylene glycol the rabbit excreted triethylene glycol in both unchanged and oxidized form. The data suggest that one of the oxidation products is a monocarboxylic acid which arises by metabolic oxidation of a single terminal hydroxyl group of the parent glycol.

Bioaccessibility (or Bioavailability) testing results:

No data.

Following the oral administration of triethylene glycol (200 mg/kg) to the rabbit, 34.3% of the dose was excreted in the urine as unchanged triethylene glycol as determined by the calorimetric method (Table 1).  In a similar experiment another animal received a 2000 mg/kg dose orally for 3 consecutive days. The calorimetric analysis gave an average value of 27.3% of the total dose excreted. By vapor phase chromatography the value was 28%.

As shown in Table 1, hydrolysis of the urine did not increase the calorimetric value for triethylene glycol determined on a chloroform solution obtained from extraction of the hydrolyzate which had been made alkaline. Triethylene glycol was chemically identified from the chloroform extract as the bis(3,S-dinitrobenzoyl) ester.

A sample of the foregoing urine which had been exhaustively extracted under alkaline conditions was acidified to pH 2 by addition of hydrochloric acid. The sample was then exhaustively extracted with chloroform. The chloroform extract was subjected to the dinitrobenzoyl calorimetric determination.  The transmittance reading corresponded to 17.3% of the administered triethylene glycol.

TABLE 1

EXCRETION OF TRIETHYLENE GLYCOL AND METABOLITES FOLLOWING ADMINISTRATION TO NEW ZEALAND WHITE FEMALE RABBITS

Animal weight (kg)	Dose (g/kg)	Per cent of dose recovered as triethylene glycol^c	Per cent of dose recovered as "hydroxyacid'
2.5	2.0^a	34.3	---
2.8	2.0^b	26.0	35.2^e
		28.6
		28.3^d

^a Urine was collected for 24 hours following administration of a single oral dose.

^b An oral dose of 2.0 g was given daily for 3 consecutive days, Urine was collected during this and a subsequent 3-day period.

^c Colorimetric determinations on chloroform extractions from alkalinized urine.

^d Urine was subjected to acidic hydrolysis prior to alkalinization and subsequent chloroform extraction.

^e Value obtained by subtracting triethylene glycol value from calorimetric determination of total alcohols in chloroform extract of acid-hydrolized acidic urine, assuming that “hydroxyacid” chromatogen possesses one-half the extinction coefficient of dihydroxy compounds such as triethylene glycol.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
After oral doses of triethylene glycol the rat and the rabbit excrete triethylene glycol in both unchanged and oxidized form. The data suggest that one of the oxidation products is a monocarboxylic acid which arises by metabolic oxidation of a single terminal hydroxyl group of the parent glycol.

Executive summary:

After oral doses of triethylene glycol the rabbit excreted triethylene glycol in both unchanged and oxidized form. The data suggest that one of the oxidation products is a monocarboxylic acid which arises by metabolic oxidation of a single terminal hydroxyl group of the parent glycol.

Reference 10

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

No data.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to guideline/s and GLP but the report contains sufficient data for interpretation of study results.

Justification for type of information:

Read across based on category approach. Please see category document for further information.

Objective of study:

other: excretion and metabolism

Qualifier:

no guideline available

Principles of method if other than guideline:

Triethylene glycol was administered by oral gavage to rats and rabbits. Urine, feces and carbon dioxide were collected and analyzed for potential metabolites.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

Randomly labeled triethylene glycol was synthesized by reaction of 14C ethylene glycol with ethylene oxide.

Species:

rat

Strain:

other: albino

Sex:

male

Details on test animals or test system and environmental conditions:

Male rats were obtained from Albino Farms, Red Bank, New Jersey.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Nonisotopic triethylene glycol was given by stomach tube to maIe albino rats in 25% W/V aqueous solution.

After receiving triethylene glycol-C14 (22.5 mg, 5.13 uc/mg, in 1 mI of water) by stomach tube, rats, varying in weight from 118 to 145 g, were housed in glass metabolism cages.

Remarks:

Doses / Concentrations:
Rats received triethylene glycol-C14 22.5 mg, 5.13 uc/mg, in 1 mI of water)

Non labeled triethylene glycol - 125, 140, 550, 600 and 790 mg/kg

No. of animals per sex per dose / concentration:

Two per dose level.

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

No data.

Details on study design:

Administration of Triethylene Glycol and Excretion by Animals
Nonisotopic triethylene glycol was given by stomach tube to maIe albino rats in 25% W/V aqueous solution. Urine was collected over sodium fluoride as runoff from metabolism cages, and all animals were allowed food and water ad libitum during the course of the experiment. Urine samples were used directly or stored frozen.

Excretion of Radioactivity by Rats
After receiving triethylene glycol-C14 (22.5 mg, 5.13 uc/mg, in 1 mI of water) by stomach tube, rats, varying in weight from 118 to 145 g, were housed in glass metabolism cages. The animals were allowed water but no food. Urine and feces were separated by a screen. Expired CO2 was collected in sodium hydroxide solution and counted at infinite thickness as barium carbonate in comparison with triethylene glycol standards at infinite thickness on talc. Dried samples of urine were counted at infinite thickness on talc. Fecal samples were oven dried at 100C and then counted at infinite thickness.

In control experiments, a 48-hour collection of urine from an untreated rat was collected in a glass metabolism cage; 0.5 ml of aqueous solution containing 11.3 mg of triethylene glycol-Cl4 was added to the urine. Air was passed through the cages for 2 days as in the metabolic experiments. Aliquots of the urine recovered from the cage by rinsing with water were dried on talc and then counted. The radioactive experiment showed the presence of 95.0%,, 99.0%, and 99.870 of the added glycol.

Fractionation of Urine after Administration of Nonisotopic Triethylene Glycol
Following administration of triethylene glycol, urine from rats was made alkaline to pH 12-13 by addition of sodium hydroxide. The urine was then exhaustively extracted with chloroform as described in the section on control studies. The chloroform extract contained triethylene glycol which was determined calorimetrically. An additional sample was then subjected to acidic hydrolysis as described in the section on control studies. The urine was then made alkaline as above for extraction of total triethylene glycol (free plus that from hydrolysis of any glucuronide ester) by chloroform. Some urine samples were acidified to pH 2-3 and then extracted with chloroform to obtain material, not extractable from alkaline solution, putative 0- (P-hydroxyethyl)oxyethoxyacetic acid, which was converted into a chromatogen by reaction with 3,.5-dinitrobenzoyl chloride for the calorimetric determination.

Vapor Phase Chromatographic Determination of an Acid Derived from the Metabolism of Triethylene Glycol
The chloroform solution obtained by a continuous exhaustive extraction (72 hours) of acidified rabbit urine obtained after administration of triethylene glycol was evaporated to dryness. The residue was transferred to a 50-ml glass-stoppered bottle with the aid of 2 ml of absolute methanol. After addition of 0.2 ml of concentrated hydrochloric acid and 2,2-dimethoxypropane (cf. Lorette and Brown, 1959), the mixture was shaken and allowed to stand at room temperature for 1 hour. An aliquot (0.2 ml) was chromatographed by the preceding procedure for vapor phase chromatographic determination of triethylene glycol in urine. The recorder response was equivalent to 1% by weight of triethylene glycol in the urine (approximately 3 times that obtained from a similar extraction of alkalinized urine). The aqueous residue from extracted alkalinized sample was then acidified to pH l-2 by addition of hydrochloric acid, The acidic solution was then exhaustively extracted with chloroform. The chloroform solution which contained no triethylene glycol was then evaporated and subjected to the esterification procedure (above). The esterified product had a retention time (8.5 minutes) on the Carbowax column which was identical to that of triethylene glycol. In comparison the dimethyl ester of ethylenedioxydiacetic acid had a retention time of 7.5 minutes. The esterified metabolite, collected from the column, and the dimethyl ester showed a similar infrared absorption spectrum. The former, however, showed a strong OH absorption at 2.9 u.

Another chloroform solution from the exhaustive extraction of the acidified urine (5 ml) was evaporated and then subjected to esterification with methanol (see above). To the residue obtained by evaporation of the esterification mixture was added 2 ml of acetic anhydride. The mixture was heated to 90” C for one-half hour. An aliquot (0.1 ml) was subjected to chromatography in the Perkin-Elmer Vapor Fractometer equipped with a column Q17 (2 meters packed with Apiezon L on Chromosorb Red). The column was adjusted to 200” C at a helium flow rate of approximately 150 ml/min. The peak at a retention time of 16 minutes, corresponding to acetylated triethylene glycol, indicated a triethylene glycol content corresponding to that obtained by chromatography of the chloroform extract of alkalinized urine. An earlier peak at 10.5 minutes differed from that obtained of an authentic sample of the dimethylester of ethylenedioxydiacetic acid (13 minutes). The peak responses of the acetylated and esterified metabolic triethylene glycol (0.6870 by weight in the urine) and that of the acetylated unmetabolized triethylene glycol of the sample (0.31% by weight in the urine) were in good agreement with the results obtained above on the Carbowax column. Evidence that triethylene glycol was determined as an acetyl derivative and that the metabolite was determined as an acetyl methyl ester was confirmed in experiments in which it was shown that neither triethylene glycol, ethylenedioxydiacetic acid, nor the methyl ester of the metabolite would pass through column Q, even when the temperature was raised to 220 C.

Fractionation of Radioactivity in Rat Urine after Oral Administration of Triethylene Glycol-Cl4
The urine plus water washings from the cage of rat no. 4 were filtered through Celite and concentrated at 37” C to a volume of 51 ml. An aliquot was made strongly alkaline by addition of ammonia water and was extracted continuously with chloroform for 3 days to obtain 59.5% of the administered radioactivity. After an additional 24-hour extraction, 0.4% additional of the administered activity was recovered. The chloroform extract was then subjected to paper chromatography on Whatman no. 1 paper with the chloroform-n-propanol ammonia system. A single radioactive zone was located on X-ray film (Rf 0.62). This substance cochromatographed with an authentic sample of triethylene glycol. The ammoniacal solution remaining from the preceding extraction was made strongly acidic by addition of concentrated hydrochloric acid and then was extracted exhaustively with chloroform to obtain 44.2% of the administered radioactivity. The chloroform solution was subjected to paper chromatography using a solvent system: 7.5 ml see-butyl alcohol, 14 ml 90% formic acid, 11 ml water (Hausman, 1952).

Investigation of Cl4 Activity in Urinary Oxalate Fraction
The urine and water washings from the cage of rat no. 5 were concentrated to a volume of 50 ml. The sample was heated at 100” C for onehalf hour after addition of 4 ml of concentrated hydrochloric acid. The sample was made alkaline with ammonia water and 1 ml of 5% aqueous calcium chloride was then added (Dakin, 1907). The resulting precipitate was washed 3 times with water and then dissolved in dilute hydrochloric acid. A precipitate was formed again by addition of ammonia water. The precipitate was again washed with water and redissolved in dilute hydrochloric acid. The solution was extracted with ether. The ethereal solution was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was dissolved in 5 ml of water and treated with ammonia water until slightly alkaline; 0.25 ml of 5%. aqueous calcium chloride was then added. The resulting fine suspension was allowed to stand for several days and then collected by centrifugation. The supernatant liquid was assayed for radioactivity in the scintillation counter. The precipitate was dissolved in 2 ml of 1 N hydrochloric acid. After radioactivity was determined, the solution was treated with 100 mg of oxalic acid dihydrate and 2 ml of concentrated hydrochloric acid at 100” C for one-half hour. A sample of the solution (0.1 ml) was removed for radioactivity determinations. The remaining solution was extracted continuously with ether for 14 hours. The ethereal solution was evaporated to dryness. The residue was dissolved in hot water. Upon cooling, two fractions of oxalic acid, m.p. 99-101” C, were obtained. These were put into solution for scintillation counting.

The supernatant liquid from the oxalate fraction was examined by descending chromatography on Whatman no. 41 filter paper in comparison with authentic samples of oxalic acid and ethylenedioxydiacetic acid with a solvent system of ethanol (4 vol) and 16% aqueous ethylamine ( 1 vol) (Long et al., 1951). The radioactive unknowns were scanned with the Actigraph II system and the reference strips were sprayed with universal pH indicator solution.

References:
HAUSMAN, W. (1952). Amino acid composition of crystalline inorganic pyrophosphatase isolated from bakers’ yeast. J. Am. Ckem. Sot. 74, 3181-3182.

LONG, A. G., QUAYLE, J. R., and STEDMAN, R. J. (1951). The separation of acids by paper partition chromatography. J. Chem. Sot., 2197-2201.

LORETTE, N. B., and BROWN, J. H., JR. (1959). Use of acetone dimethyl acetal in preparation of methyl esters. J. Org. Ckem. 24, 261.262.

Details on dosing and sampling:

After receiving triethylene glycol-C14 (22.5 mg, 5.13 uc/mg, in 1 mI of water) by stomach tube, rats, varying in weight from 118 to 145 g,

Statistics:

No data.

Type:

excretion

Results:

86.1 - 94.0% of the administered dose was recovered within 5 days in the urine. In two of four rats examined, >85% of the administered dose was recovered within one day.

Details on absorption:

No data.

Details on distribution in tissues:

No data.

Metabolites identified:

yes

Details on metabolites:

After oral doses of triethylene glycol the rat and the rabbit excrete triethylene glycol in both unchanged and oxidized form. The data suggest that one of the oxidation products is a monocarboxylic acid which arises by metabolic oxidation of a single terminal hydroxyl group of the parent glycol.

Bioaccessibility (or Bioavailability) testing results:

No data.

A series of paired rats were given triethylene glycol orally in doses ranging from 125 to 600 mg/kg (Table 1). The values for triethylene glycol recovered in the 24-hour urine samples were 27.4 to 66%. The calorimetric data on extracts of acidified urine again showed enhanced values consistent with the possible presence of a hydroxy acid from the metabolism of triethylene glycol.

Following oral administration of triethylene glycol-C14, the rat (Table 2) showed a consistently high elimination of C14 activity, the urine serving as the major excretory route in all cases. At the end of a S-day period, the total recovery of C14 was 91-98s of the administered dose. Approximately 95% of the recovered activity, or 90% of the C14 dose, appeared in the urine. A lesser amount appeared in the feces and an indeterminate amount of fecal radioactivity arose through contamination by urine.  Respiratory carbon dioxide afforded, in the 4 rats studied, elimination of only 1% of the activity in the dose of glycol, and most of this activity appeared during the first 12-hour period after administration (Table 3).

Similarly, the major urinary excretion of radioactivity appeared during the period of the first day following administration. The nature of the urinary radioactivity was approached by a variety of chemical and physical techniques.

The chromatographic examination of products coprecipitated with calcium oxalate from the urine of rat no. 5 yielded in addition to material with Rf corresponding to oxalic acid (origin to Rf 0.35) two other acidic components, Rf 0.37 and Rf 0.48-0.57. The major component at Rf 0.37 was not identified. The Rf value of authentic ethylenedioxydiacetic acid (“ethylene bis(glycolic)” acid) was Rf 0.50. The combined radioactivity of all the components including oxalate was 0.0046% of the administered radioactivity. Following addition of carrier oxalic acid and several recrystallizations, the radioactive values indicated that at most only 3% of the above (0.00017, of administered radioactivity) was present as oxalic acid.

Fractions from the urine of rat no. 4 were subjected to chromatographic study by a different procedure, which served again to indicate the acidic nature of some of the metabolites arising from the metabolism of triethylene glycol and the absence of appreciable quantities of the lower glycols, diethylene glycol and ethylene glycol, which are considered to be physiologically undesirable. Chromatography and autoradiography of the chloroform solution obtained by continuously extracting the alkalinized urine revealed the presence of one radioactive zone, corresponding in Rf value and cochromatography to authentic triethylene glycol. A chloroform solution, obtained subsequently by continuously extracting aqueous residue of acidic pH, contained two radioactive zones, Rf 0.64 and Rf 0.84, upon paper chromatography in the set-butyl alcohol-formic acid-water system. The zone at Rf 0.84 was not identified. Authentic ethylenedioxydiacetic acid chromatographed at Rf 0.64 under similar conditions.

Table 1 EXCRETION OF NONISOTOPIC TRIETHYLENE GLYCOL BY MALE ALBINO RATS

FOLLOWING ORAL ADMINISTRATION

			Colorimetric values for % of dose recovered in chloroform extracts.
Pair No.^a	Weight of pair (g)	Total dose (mg/kg)	Alkaline urine	Alkaline urine after acidic hydrolysis	Acidic urine after acidic hydrolysis
1	360	125	66.0	67.2	---
2	320	140	65.0	70.8	---
3	450	550	37.8	44.6	56.7^c
4	590	600	27.4^b	---	40.3
5	320	790	---	---	60.0 51.7

^a Two rats of approximately equal weight were used for each pair.

^b The value obtained by vapor phase chromatography was 30.8%.

^c Value for “hydroxyacid” = (56.7-44.6) X 2 = 24.2% of dose.

TABLE 2 EXCRETION OF TRIETHYLENE GLYCOL AND METABOLITES BY MALE ALBINO RATS FOLLOWING A SINGLE ORAL DOSE (22.5 MG) OF RANDOMLY LABELED TRIETHYLENE GLYCOL-C¹⁴

		Radioactivity recovered in 5 days as % of administered dose
Rat No.	Weight (g)	Expired air	Urine	Feces	Total
2	118	1.2	89.0	5.3	95.5
3	120	0.9	94.0	3.4	98.3
4	112	0.8	93.5^a	2.0	96.3
5	145	0.9	86.1^b	3.6	90.6

^a Daily urine values: Day 1 = 92.7; 2 = 0.53; 3 = 0.21; 4 plus 5 = 0.09.

^b Daily urine values: Day 1 = 85.2 ; 2 = 0.47 ; 3 = 0.29; 4 plus 5 = 0.15.

TABLE 3 ELIMINATION OF TRIETRYLENE GLYCOL AS RESPIRATORY CARBON DIOXIDE

	% of Dose as C^14O2
Rat No.	0 -12 Hr	12 -24 Hr	24 -36 Hr	36 -48 Hr	48 -60 Hr	Total
2	0.81	0.10	0.06	0.04	0.17	1.18
3	0.52	0.09			0.31 (24 - 60 hours)	0.91

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
The major part of the radioactivity appeared in the urine. The data from fractionation of the urine indicated that only negligible quantities (if any) of Cl4 were present as oxalic acid.

Executive summary:

After oral doses of triethylene glycol the rat and the rabbit excrete triethylene glycol in both unchanged and oxidized form. The data suggest that one of the oxidation products is a monocarboxylic acid which arises by metabolic oxidation of a single terminal hydroxyl group of the parent glycol. After oral administration of triethylene glycol-C¹⁴ the rat eliminated only trace quantities of C^l4 activity as respiratory carbon dioxide. A small but measurable amount of radioactivity was found in the feces. The total elimination of radioactivity (in urine, feces, and expired air) during the 5-day period following a single oral dose (22.5 mg) was 91-98%. The major part of the radioactivity appeared in the urine. The data from fractionation of the urine indicated that only negligible quantities (if any) of C^l4 were present as oxalic acid. The major metabolic products had properties which suggested that triethylene glycol is degraded by the route: HOCH₂CH₂OCH₂CH₂OCH₂CH₂OH ---> HOCH₂CH₂OCH₂CH₂OCH₂COOH ---> HOOCCH₂OCH₂CH₂OCH₂COOH The foregoing data, showing a high degree of elimination of triethylene glyco] and its metabolites by way of the urine, is consistent with many findings pointing to the low or limited toxicity of triethylene glycol.

Reference 11

Endpoint:

dermal absorption in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read across based on category approach. Please see category document for further information.

Principles of method if other than guideline:

Occuded dermal exposure to DEG was on the clipped backs of rats. Breath, urine, feces, and blood was collected and radioactivity measured. Urine was analyzed for DEG and its metabolites by HPLC.

GLP compliance:

no

Specific details on test material used for the study:

DEG was purchased from Aldrich Chemical Co, Inc. (Milwaukee, WI). [U-14C]-DEG, specific activity 14 mCi/mmol, was obstained from Amersham Laboratories (Buckinghamshire, UK) and was further purified by HPLC to a radiochemical purity of 98%.

Radiolabelling:

yes

Remarks:

[U-14C]-DEG

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

Adult male Fischer 344 rats (220-280 g) were purchased from Charles River Breeding Laboratories, Inc. (Raleigh, NC). All animals were quarantined at least 1 week before they were used. Rats were fed Certified Purina Rodent Chow #5002 and furnished tap water ad libitum. During experiments rats were housed individually in glass metabolism chambers which provide for separate collection of urine, feces, and breath.

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Doses:

The dermal dose formulation contained ~25 microCi of radiolabel and 50 mg of unlabeled DEG per animal.

No. of animals per group:

3-5

Details on study design:

Dermal doses were administered onto a 12-cm2 area of skin in the intrascapular area on the backs of rats. Hair had been clipped from the area the previous day, and each rat was inspected to make sure the skin had not been nicked. After dosing, a nonocclusive protective appliance was glued over the dose area.

Determination of Radioactivity. Radiolabeled components eliminated in breath were collected separately by drawing air through a cold trap (-60°C) containing 100 ml of ethanol, and then through two traps, each containing 400 ml of 1 N NaOH. Aliquots of urine and trapping solution from the breath traps were added directly to vials containing scintillation cocktail (Scintiverse E, fisher Chemical Co.). Samples of tissue, feces, and blood (0.2-0.3 g) were digested in Soluene-350 (2 ml; Packard Instrument Co.). After digestion, samples requiring bleaching were decolorized with perchloric acid/hydrogen peroxide prior to addition of scintillation cocktail (Scintiverse E). Samples containing base and scintillate (e.g., sodium hydroxide. sodium carbonate, or Soluene-350) were kept in the dark overnight before they were assayed by liquid scintillation spectrometry.

Analysis of Biological Samples by HPLC. Urine was analyzed by HPLC for unchanged DEG and its metabolites.

Metabolites were isolated and identified by HPLC and NMR.

Absorption in different matrices:

Dermally administered DEG (50 mg/12cm2) was slowly and steadily absorbed. Approximately 3% of the applied dose was absorbed per day and excreted in the urine over a 72 hour period. A total of 9.1 +/- 1.5% of the dose was recovered in the excreta, and 0.9 +/- 0.3% was recovered in tissues.

Time point:

72 h

Dose:

50 mg / 12 cm2

Parameter:

percentage

Remarks:

recovered in excreta

Absorption:

9.1 %

Conclusions:

DEG slowly penetrated the skin of rats after application of 50 mg to a 12 cm2 area. Only about 10% of the dose was absorbed in 72 hr of exposure and the absorbed dose appeared to have the same fate as doses administered iv or orally.

Executive summary:

DEG slowly penetrated the skin of rats after application of 50 mg to a 12 cm2 area. Only about 10% of the dose was absorbed in 72 hr of exposure and the absorbed dose appeared to have the same fate as doses administered iv or orally.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

Absorption:

Higher ethylene glycols are completely and rapidly absorbed in animals from the gastrointestinal tract due in part to their complete solubility in water and low molecular weights. Likewise, the absorption estimate via inhalation route will be assumed as 100 percent. For dermally applied DEG, 47.2, 32.6, 29.2 and 16.8% remained after 15 minutes, 64 minutes, 8 hours and 24 hours, respectively. After 15 minutes of dermal contact with DEG, a radioactive peak appeared at ~ 112 microns into the layer of the skin indicating rapid absorption via dermal route. After 8h and 24h contact, 11.9% and 32.2% radioactivity was detected in the rat urine.

Distribution, Bioaccumulation Potential:

Since ethylene glycol substances are completely soluble in water, they are expected to be well distributed throughout the aqueous tissues of the body and have no or low concentrations in adipose tissue. Therefore, ethylene glycol analogues are considered to have no bioaccumulation potential. For DEG, the apparent volume of distribution was determined at 1 ml/g; DEG and its metabolites did not accumulate in tissues.

Metabolism:

The higher ethylene glycols are linked by stable ether bonds and are not extensively metabolized in the body. The main pathway for metabolism of the ethylene glycol analogues is oxidation via alcohol dehydrogenases and aldehyde dehydrogenases however, the majority of ethylene glycol analogues will be either unchanged or conjugated with glucuronide. The potential for metabolic oxidation of ethylene glycols decreases with increasing number of oxyethylene units per molecule as seen experimentally from in vitro derived Vmax and Km kinetic values.

In rat, oxalic acid is not a significant DEG metabolite and metabolism becomes saturated at higher doses. In both the rat and the rabbit, TEG showed a high degree of urinary excretion of unchanged compound, in addition to small amounts of monocarboxylic acid arising by metabolic oxidation of the terminal hydroxyl group on the parent glycol as well as glucuronide conjugation. The results in vivo agree with DEG and TEG agree with the in vitro metabolism data via aldehyde dehydrogenase, indicating the decreased potential for metabolic oxidation with increasing number of oxyethylene repeat units per molecule. Based on in vitro data, TTEG is expected to be oxidized to a lesser extent than TEG and either excreted unchanged or as glucuronide conjugates of the parent glycol. The low order of excretion of radioactivity by way of respiratory carbon dioxide demonstrated that the cleavage of carbon bonds of the administered TEG occurred to only a limited extent. Respiratory carbon dioxide in the rat constituted only 1% of the radioactive dose and most of this activity appeared during the first 12-hour period after administration. The evidence suggests that metabolic cleavage of the ether linkages and formation of ethylene glycol wound not be important events in the metabolism of TTEG.

Excretion:

The excretion of ethylene glycols is rapid with the urine being the major excretory route in the rat and rabbit. Animal studies indicate a high degree of urinary excretion of unchanged TEG in addition to the excretion of oxidized glycol acid metabolite. >85% of the orally administered TEG to rats was recovered within 24 hours. 86.1 - 94.0% of the administered dose was recovered within 5 days in the urine. Approximately 95% of the recovered radioactivity, or 90% of the C14 dose, appeared in the urine with lesser amount in the feces. The total elimination of radioactivity (in urine, feces, and expired air) during the 5-day period following a single oral dose in rat was 91-98%. For dermally applied DEG, in contact with the rat skin for 8 and 24 hours, 12 and 32.2% of the radioactivity was identified in the urine, respectively.