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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
received: 23 July 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study similar to OECD guideline 417. Minor restrictions in reporting.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
purchased from Fluka Chemical Corp. (NY, USA9
Purity: > 99.5%

[1,2-Ethylene-14C]bis(2-methoxyethyl) ether:
synthesized by Pathfinder, Inc. (MO, USA)
Purity: 99%
Specific activity: 0.88 mCi/mmol
Radiolabelling:
yes
Remarks:
[1,2-Ethylene-14C]bis(2-methoxyethyl) ether

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc (MA, USA)
- Weight at study initiation: 190-220 g (metabolism study); 190-240 g (toxicity study)
- Fasting period before study: 18 hours prior to treatment (metabolism study)
- Housing: five per cage (toxicity study)
- Individual metabolism cages: yes, Roth-type glass metabolism cage (individually; metabolism study)
- Diet (e.g. ad libitum): NIH-07 rat and mouse diet (Ziegler Brothers, Inc., PA, USA), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum; distilled water (metabolism study)
- Acclimation period: 3 days (metabolism study)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24°C
- Humidity (%): 45-51 %
- Photoperiod (hrs dark / hrs light): 12/12

Metabolism study:
Laboratory air, from which moisture, carbon dioxide, and organic vapors had been removed, was drawn by vacuum through the metabolism chambers at a rate of 500 mL/min.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
a. Metabolism study:
A dosing was prepared for the metybolism study dissolving appropriate amounts of NIH-07 rat and mouse diet [1,2-Ethylene-14C]bis(2-methoxyethyl) ether and Bis(2-methoxyethyl) ether in distilled water to give a final concentration of 1.02 mmol/mL (specific activity 29 µCi/mmol). A second dosing was prepared by diluting sufficient [1,2-Ethylene-14C]bis(2-methoxyethyl) ether in distilled water to give a final concentration of 0.0102 mmol/mL (specific activity 980 µCi/mmol). Radioactivity was quantified by scintillation spectrometry.

b. Toxicity study:
For the toxicity study separate dosing solutions of 2-(2-Methoxyethoxy)ethanol and (2-Methoxyethoxy)acetic acid were prepared in distilled water to give each a final concentration of 1.02 mmol/mL.
Duration and frequency of treatment / exposure:
a. Metablism study: single dose (gavage)
b. Toxicity study: 20 applications (one daily; gavage)
Doses / concentrations
Remarks:
Doses / Concentrations:
a. 5.1 or 0.051 mmol/kg bw; 148 or 50 µCi/kg bw
b. 5.1 mmol/kg bw/d
No. of animals per sex per dose:
a. 5
b. 50
Control animals:
not specified
Positive control:
None
Details on study design:
a. Metabolism study:
Immediately following the administration of the individual doses, the rats were returned to their cleaned individual metabolism chambers for collection of exhaled compounds, urine and feces. The emergent air from each chamber was drawn by vaccum first through a sorbent air-sampling tube containing an 800-mg front section and a 200-mg back section of coconut-based charcoual to trap exhaled organic compounds and subsequently through an absorption lower filled with 500 mL of Ethanolamine to trap expired carbon dioxide. the air-sampling tubes and the Ethanolamine solutions were changed periodically as required to avoid loss of radioactivity. Urine was collected at 6, 12, 24, 48, 72 and 96 hours. Each urine sample was diluted to 10 or 25 mL and immediately passed through an Acrodisc filter having a 0.45 µm pore size. Feces were collected at 24, 48, 72 and 96 hours. The air-sampling tubes, uribe and feces were stored at -20°C until analysed. The rats were killed by asphyxiation with carbon dioxide 96 hours after dosing. The cages were washed successively with water and Methanol, and the washes from each cage were collected separately for 14C analysis.

b. Toxicity study:
Rats were randomly assigned to three groups of 50 animals each and were housed five per cage. All animals within one group were administered by gavage a daily dose for up to 20 consecutive days of 2-(2-Methoxyethoxy)ethanol or (2-Methoxyethoxy)acetic acid at 5.1 mmol/kg bw, a molar dose equivalent to that of Bis(2-Methoxyethyl) ether previoulsly shown to produce testicular atrophy. Animals in each treatment group were killed in groups of five at 2-day intervals on Days 2 through 21 for evaluation of any histological changes in the testis. At necropsy animals were anesthetized with Secobarbital sodium and killed by exsanguination. The testes were excised, weighed separately, and fixed in Bouin's solution for 24 hours. The testes were then rinsed with 70% Ethanol and embedded in paraffin wax. The tissues were sectioned at 5 µm and stained with periodic acid Schiff stain for demonstration of the spermatid acrosome or hematoxylin by light microscopy.
Details on dosing and sampling:
Determination of radioactivity:
Aliquots of the diluted urine samples were added to liquid scintillation vials containing 3 mL of Methanol and 10 mL of scintillation medium for the determination of 14C. Aliquots of the cage washes were dissolved in 10 mL of the same scintillation medium. Front and back sections oof charcoal from each air-sampling tube were desorbed in separate 5 mL-quantities of Bis(2-methoxyethyl ether, a solvent found to desorb greater than 98 % of [1,2-Ethylene-14C]bis(2-methoxyethyl) ether adsorbed in amounts typical of those collected from the emergent chamber air. A 1-mL aliquot was transferred to a liquid scintillation vial containing 10 mL of the scintillation medium. The carcass digests were homogenised separately with Polytron Nodel PCU-1 homogenizer equipped with a PC-10 probe, and 1-mL aliquots were anallysed in the scintillation medium. Aliquots of the Ethanolamine were prepared for scintillation couting in Permaflour V. Feces, while still frozen were ground with equal weight of microcrystalline cellulose in a Model A10 microanalytical mill until a homogeneous mixture was obtained. Weighed portions of feces-cellulose mixtures were burned in a sample oxidizer.The 14CO2 from the combustion was absorbed in 6 mL of Carbo-Sorb and analysed in Permafluor V. The radioactivty in the samples was measured in a model LS8100 liquid scintillation spectrometer. Counting efficiencies were determined by external standard method.

Separation, quantification and isolation of urinary metabolites:
Prior to analysing the urine by HPLC, suitable conditions for the seapartion of compounds representing possible metabolites of Bis(2-methoxyethyl ether were established. The HPLC system consisted of a Model ALC/GPC 201 liquid chromatograph equipped with two Model 6000A pumps controlled by a Model 720 system controller. Samples were introduced by a Waters WISP 710A automatic injector module, and eluted compounds were detected by a Model 1040A spectrophotometric detector set at 210 nm or by a Tri-Carb RAM 7500 radioacitivty monitor equipped with 50-µL RAM 754 heterogeneous flow cell. Better than base-line resolution for all compounds wass achieved using a 50-cm long x 9 mm i.d. stainless-steel column packed with 10-µm Partisil-10 OD-2 in series with a 15-cm long x 3.9-mm i.d. stainless-steel column packed with 5-µm spherical NOVA-PAK C18. The column temperature was maintained at 30!C using a column heater module and an associated temperature control module. Radioactive urinary components were chromatographed with 1% acetic acid in water isocratically for 20 min, then with a Methanol: 1% acetic acid solvent system programmed from 15 to 80% Methanol using a linear gradient over a 40 min period. The flow rate was maintained at 1 mL/min. Each urine sample was thawed, and a 3-mL portion was acidified to pH 2 with H2SO4. Aliquots of the acidified urine samples were injected into the HPLC, and radioactive components were detected and quantified by the radioactivity monitor. The retention time of each radioactive component was recorded, and the radioctive fractions were collected separately using a Model 2103 fraction collector for further characterisation of the radioactive compounds.

Mass spectral identification of urinary metabolites:
Each radioactive fraction of urine, collected from the HPLC column, was lyophilized using a Model 75035 freeze-dryer, and the residue was redissolved in a small amount of Methanol for analysis by gas-chromatograph - mass spectrometry (GC-MS).

Radioactive constituent of breath:
Volatile organic compounds expired by two rats during the 24-hour period immediately following the administration of Bis(2-methoxyethyl) ether at 5.1 mmol/kg bw were collected using a single, charcoal air-sampling tube for each rat. The charcoal sections from these sampling tubes were combined and subsequently desorbed for 24 hours with 10 mL of Methanol. aliquots of the Methanol solutions were analysed by HPLC and GC-MS to characterise any radioactive component eliminated in the breath.
Statistics:
Statistical differences between group means were determined using one-way analysis of variance. The level of significance chosen was p<0.05.

Results and discussion

Preliminary studies:
No data

Toxicokinetic / pharmacokinetic studies

Details on absorption:
n.a., metabolism/excretion study
Details on distribution in tissues:
n.a., metabolism/excretion study
Details on excretion:
Please refer to "Any other information on results"

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Please refer to "Any other information on results"

Any other information on results incl. tables

Excretion of radioactivity

In rats given a single dose of [1,2-Ethylene-14C]bis(2-methoxyethyl) ether, the elimination of the administered radioactivity occured principally through the kidneys. The recovery of the administered 14C directly from this route over 96 hours was 85.7 +/- 1.7% for rats treated with the high dose (5.1 mmol/ kg bw). When the amounts of 14C in the cage washes - radioactivity considered to be from residual urine- were included, the total recoveries from the urinary route were 86.5 +/- 1.6% for the high dose and 90.0 +/- 1.2% for the low dose. The elimination of radioactivy in the urine was rapid with 77.5 +/- 3.1% of the high dose and 83.1 +/- 2.7% of the low dose excreted over the first 24 hours. Only an additional 9.0% of the high-dose 14C and 6.9% of the low-dose 14C were excreted during the ensuing 72 hours. Respired 14CO2 collected from rats receiving either 5.1 or 0.051 mmol/kg bw doses of Bis(2 -Methoxyethyl) ether was negligible, amounting to only 1.4 +/- 0.1 and 3 .6 +/- 0.3% of the administered dose, respectively. Only trace amounts of 14C were found in the breath as volatile organics, totaling 1.2 +/- 0.1% of the administered high dose and 0.4 +/- 0.1% of the low dose. Less than 5% of the administered 14C was excreted in the feces. At 96 hours, the elimination of 14C was nearly complete with less than 2.5% of the dose remaining in the carcasses.

Table 1: Recoveries of14C 96 Hours after Oral Application of Radiolabelled Diglyme (Bis(2-methoxyethyl) ether) to Male Sprague-Dawley Rats

 

% of Administered Bis(2-methoxyethyl) ether

Sample

Low dose1

High dose2

 

 

 

Total urinary

90.0±1.2

86.5±1.6

  Urine

88.8±1.4

85.7±1.7

  Cage wash

1.2±0.3

0.8±0.1

 

 

 

Carbon dioxide

3.6±0.3

1.4±0.13

 

 

 

Volatile organic

0.4±0.1

1.2±0.13

 

 

 

Feces

2.9±0.6

4.6±1.03

 

 

 

Carcass

1.7±0.2

2.2±0.4

 

 

 

Total recovery

98.6±0.8

95.9±1.3

 

 

 

1             0.051 mmol/kg bw (980 µCi/mmol); mean of 5 rats

2             5.1 mmol/kg bw (29 µCi/mmol); mean of 5 rats

3             p < 0.05 by comparison with corresponding low-dose value

Table 2:Bis(2-Methoxyethyl) Ether – 96 Hours Urinary Metabolic Profile (Calculated as Percentage of Dose)

Metabolite

Low dose1

High dose2

 

 

 

(2-Methoxyethoxy)acetic acid

70.3±1.3

67.9±3.3

Diglycolic acid

6.7±1.0

3.9±1.0

Methoxyacetic acid

5.8±0.8

6.2±0.8

2-Methoxyethanol

2.2±1.1

0.8±0.3

Bis(2-methoxyethyl)ether

0.4±0.1

1.8±0.33

2-(2-Methoxyethoxy)ethanol

0.3±0.2

<0.1

N-(Methoxyacetyl)glycine

0.1±0.1

0.3±0.2

 

 

 

1             0.051 mmol/kg bw (980 µCi/mmol), mean of 5 rats

2             5.1 mmol/kg bw (29 µCi/mmol); mean of 5 rats

3                 p < 0.05 by comparison with corresponding low-dose metabolite value

Identification of the radioactive component in expired air

Volatile organic compoinds collected from the breath were desorbed from the charcoal sampling tubes with Methanol and analysed by HPLC. The single radioactive component gave a retention time identical to that of authentic Bis(2 -methoxyethyl ether. The identity of this compound was confirmed by matcihng the GC retention time and mas spectrum with those of Bis(2 -methoxyethyl) ether.

Histopathology

Examination of the testes of rats given as many as 20 consecutive daily doses of either primary metabolite, (2 -Methoxyethoxy)acetic acid, or its metabolic precursor, 2 -(2 -Methoxyethoxy)ethanol), at 5.1 mmol/kg bw/d, indicated that no degenerative changes had occured.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The principal metabolites of Diethylene glycol dimethyl ether after a single dose of 5.1 or 0.051 mmol/kg bw to male rats are (2-Methoxyethoxy)acetic acid and Methoxyacetic acid. Within 96 hours approximately 86-90% of the administered dose was excreted via urine. Neither (2-Methoxyethoxy)acetic acid nor 2-(2-Methoxyethoxy)ethanol induced gross or microscopic abnormalities in testes of rats dosed with 5.1 mmol/kg bw/d for 20 consecutive days.
Executive summary:

Male rats were treated with a single oral dose of Diethylene glycol dimethyl ether and the urine, feces and expired air were examined for the test item and its metabolites. 2-(2-Methoxyethoxy)ethanol, the principal metabolite of Diethylene glycol dimethyl ether, was administered to male rats on 20 consecutive days. After study termination the rats were examined for testicular lesions.

The principal metabolites of Diethylene glycol dimethyl ether after a single dose of 5.1 or 0.051 mmol/kg bw to male rats are (2-Methoxyethoxy)acetic acid and Methoxyacetic acid. Within 96 hours approximately 86-90% of the administered dose was excreted via urine. Neither (2-Methoxyethoxy)acetic acid nor 2-(2-Methoxyethoxy)ethanol induced gross or microscopic abnormalities in testes of rats dosed with 5.1 mmol/kg bw/d for 20 consecutive days