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Basic toxicokinetics

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basic toxicokinetics in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
received: 13 October 1992
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline study following a scientifically sound study design.

Data source

Reference Type:
Report Date:

Materials and methods

Objective of study:
Test guideline
no guideline available
not applicable
Principles of method if other than guideline:
Human and rat microsomes were incubated with Diethylene glycol dimethyl ether. The formation of 2-Methoxyethanol was determined.
GLP compliance:
not specified

Test material

Details on test material:
Supplier: Fluka Chemical Corp. (NY, USA)
Radiolabelled Diglyme [1,2-Ethylene-14C]bis (2-methoxyethyl) ether with a specific activity of 29.8 mCi/mmol was acquired from Chemsyn Science Laboraties (KS, USA)
[1,2-Ethylene-14C]bis (2-methoxyethyl) ether

Test animals

Details on test animals and environmental conditions:
- Source: Charles river Breeding Laboratories, Inc. (MA, USA)

Phenobarbital (PB) was administered by i.p. injection at a dose of 100 mg/kg bw/d for 3 days before they were killed. ß-Naphthoflavone (ß-NF), suspended in corn oil, was injected i.p. for 3 days at dose of 80 mg/kg bw/d. Rats were given drinking water containing 15% (v/v) Ethanol for 3 days before the isolation of microsomes. Diglyme treatment consisted of providing animals with drinking water conatining 0.6% (w/v) dyglyme for 4 days. Based on average consumption of the diglyme solution, rats were given a daily dose of diglyme of about 700 mg/kg bw during the course of the induction. All diiglyme and ethanol drinking water solutions were prepared fresh daily and provided ad libitum.
Animals used in these experiments weighed between 160 and 230 g when they were killed.

Rats were asphyxiated with carbon dioxide. Livers were immediately excised, rinsed and then homogenized in ice-cold 0.25 M Sucrose containing 0.125 M Tris-HCl (pH 7.4) and 1 mM Ethylenediaminetetraacetic acid (EDTA). Homogenates were centrifuged at 15,000 g for 15 min at 4°C, and microsomes were pelletd from the resulting supernatant by centrifuging at 100,000 g for 1 hour. Microsomes were washed to remove cytosolic contamination by resuspending the pellet in 0.05 M Potassium phosphate buffer (pH 74.) and centrifuging at 100,000 g.
Human hepatic microsomes were acquired from Human Biologics, Inc. (AZ, USA). Microsomes isolated from the human B-lymphoblastoid AHH-a cell line and expressing human P-450s were purchased from Genteat Corp. (MA, USA). These microsomal preparations were assayed for specific P-450-catalysed enzyme activities, and in most cases, the measured specific activities were within the range expressed by human hepatic microsomal preparations. All microsomes were stored at -80°C before use.

Administration / exposure

Route of administration:
other: Diglyme was added to the microsomal fraction
physiological saline
Details on exposure:
Metabolic assay
Microsomal incubations were conducted at 37°C in 0.05 M Potassium phosphate (pH 7.4), 5 mM MgCl2 buffer and contained 1 mM Diglyme. The NADPH-generating system consisted of the following: 10 mM Glucose-6-phosphate, 1.3 U Glucose-6-phosphate dehydrogenase and 0.5 mM NADP+. Blanks contained no NADP+. When radiolabelled Diglyme was added to the incubations, the final specific activity was diluted to 6 mCi/mmol. Unless otherwise indicated, all Diglyme incubations contained 1 mg microsomal protein and were terminated after 30 min by addition of phosphoric acid to a final concentration of 55 mM.

Biochemical assay
Pentoxyresorufin dealkylase and NADPH-cytochrome (cyt) c (P-450) reductase activity were measured. Incubations of aniline hydroxylase activity and ethoxycoumarin deethylase activity were performed in 0.05 M Potassium phosphate (pH 7.4), 5.0 mM MgCl2 buffer and contained the NADPH-generating system decribed for Diglyme incubations. The 30 min Aniline hydroxylase incubations contained 5.0 mM Aniline. The product of Aniline hydroxylase activity, p-Aminophenol and Ethoxycoumarin deethylase activity, were measured. P-450 content was determined spectrophotometrically.
Duration and frequency of treatment / exposure:
Single application, 30 min incubation
Doses / concentrations
Doses / Concentrations:
1 mM Diethylene glycol dimethyl ether (Diglyme)
No. of animals per sex per dose:
n.a., in vitro study
Control animals:
other: n.a., in vitro study
Positive control:
Details on study design:
Please refer to "Details on exposure"
Details on dosing and sampling:
Please refer to "Details on exposure"
One-way analysis of variance was performed using Statgraphics, version 5 (STSC Inc. MD, USA) statistical package. Differences between untreated controls and specific treatments were determined using Scheffe's test for multiple comparisons. Least square linear regression and Student's t-test were performed to evaluate the strength of association between two variables.

Results and discussion

Preliminary studies:
No data

Toxicokinetic / pharmacokinetic studies

Details on absorption:
n.a., metabolism study
Details on distribution in tissues:
n.a., metabolism study
Details on excretion:
n.a., metabolism study

Metabolite characterisation studies

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

Any other information on results incl. tables

NADPH-dependent metabolism of Diglyme by rat hepatic microsomes

Diglyme metabolites were determined using radiolabelled Diglyme and HPLC analysis. Three major metabolites were detected in rat hepatic microsomal incubations containing an NADPH-generating system. Two of the metabolites were identified as 2 -Methoxyethanol and 2 -(2 -Methoxyethoxy)ethanol. The identity of the third metabolite was not determined.Analysis of incubations containing rat hepatic microsomes from untreated rats, an NADPH-generting system and 1 mM Diglyme indicate less than 2% of the added Diglyme was metabolized after 30 min.

Metabolism of Diglyme by human hepaic microsomes

Human hepatic microsomes obtained from eight subjects were assayed for Aniline hydroxylase activity. The mean value for the amount of Aniline metabolized/min/nmol P-450 for humans was 0.60. Haevy alcohol use was reported in the case histories of two of the subjectes. the Aniline hydroxylase activities measured in microsomes isolated from these subjects were equal to or greatr than the mean but were not substantially elevated over the other measured values.

Human hepatic microsomes catalysed the NADPH-dependent cleavage of Diglyme to 2 -Methoxyethanol. In addition to 2 -Methoxyethanol, 2 -(2 -Methoxyethoxy)ethanol and an unknown metabolite were also identitfied in human microsomal incubations containing radiolabelled Diglyme. Formation of these products was demonstrated to be NADPH-dependent. These productes were also identified with rat hepatic microsomes containing an NADPH-generating system. A mena value of 19.7 nmol 2 -Methoxyethanol/min/nmol P-450 was determined for the 30 min incubations with human microsomes.

A test of the association between the Aniline hyroxylase activity (nmol/min/mg protein) of human microsomes and the amount of 2 -Methoxyethanol (nmol/mg protein) formed by these microsomes during a 30 min incubation yielded a correlation coefficient of 0.87. In a similar manner, the combined Aniline hydroxylase and the 2 -Methoyethanol results from untreated and Ethanol-, Phenobarbital-, diglyme- and ß-Naphthoflavone-preteated rats yielded a correlation coefficient of 0.71. The association between these two viariables for human and rat hepatic microsomes was statistically significant (p<0.01).

Effects of P-450 induces on 2 -Methoxyethanol formation (rat microsomes)

Treatment of rats with drinking water containing Diglyme before they were killed significantly increased microsomal levles of P-450 (p<0.05). P-450 levels increased by 70% above control values. Diglyme pretreatment also significantly increased P-450 -associated enzyme activities. Statisitcally significant increases were noted in the specific activity levels of Ethoxycoumarin deethylase and pentoxyresorufin dealkylase following Diglyme pretreatment. Pretreament of animals with either Ethanol or Phenobarbital augmented the metabolism of Diglyme to 2 -Methoxyethanol catalysed by rat hepatic microsomes. Both the ethanol- and phenobarbital-induced increases were determined significant regardless of whether the amount of 2 -Methoxyethanol formed was expressed per mg protein or per nmol P-450. Pretreatment of animals with Diglyme also significantly (p<0.05) increased the capacity of microsomes to convert Diglyme to 2 -Methoxyethanol.

Time- and protein-dependent formation of 2 -Methoxyethanol (rat microsomes)

The effects of incubation time on the cleavage of Diglyme to 2 -Methoxyethanol by phenobarbital-induced rat hepatic microsomes were examined. 2 -Methoxyethanol formation was time dependent, and measured rates of formation for 2 -Methoxyethanol were similar over the first 30 min of the experiment (approx. 0.5 nmol/min). After 30 min, rates of 2 -Methoxyethanol formation appeared to diminish slightly with increasing incubation time. The amount of Diglyme cleaved to 2 -Methoxyethanol during 30 min incubations with phenobarbital-induced microsomes was also dependent on the concentration of microsomal protein present in the incunation media. Increasing concentrations of microsomal protein produced proportionally greater amounts of 2 -Methoxyethanol up to about 1 mg protein/mL. Additions of microsomal protein above 1.5 mg/mL did not increase the amount of 2 -Methoxyethanol formed during 30 min incubations.

Effects of P-450 inhibitor on 2 -Methoxyethanol formation

Metyrapone (inhibitor of phenobarbital-induced P-450), TAO (specific inhibitor of P450 III) and Isoniazid (inhibitor of P450 IIIEI) significantly reduced the cleavage of Diglyme to 2 -Mthoxyethanol catalysed by phenobarbital-induced microsomes. Isiniazid produced a 67 % inhibition of 2 -Methoxyethanol formation in phenobarbital-induced microsomes, while Metyrapone and TAO produced inhibitions of 34 and 51 %, respectively. Only Isoniazid significantly reduced the cleavage of Diglyme to 2 -Methoxyethanol catalysed by microsomes isolated from ethanol- or diglyme-pretreated rats. Isoniazid inhibited 2 -Methoxyethanol formation by 85 % with ethanol-induced microsomes and by 53 % with microsomes isolated from diglyme-pretreated rats.

Applicant's summary and conclusion

Interpretation of results (migrated information): other: Diglyme is metabolised to 2-Methoxyethanol and 2 -(2 -Methoxyethoxy)ethanol
Metabolism of Diglyme (Diethylene glycol dimethyl ether) to 2-Methoxyethanol and 2 -(2 -Methoxyethoxy)ethanol by human and rat microsomes is NADPH-dependent and pretreatment of rats with the P-450 inducers Phenobarbital and Ethanol significantly increased the microsomal metabolism of Diglyme. The central ether linkage of Diglyme is cleaved by both the human and rat orthologues of P450 IIEI. Due to the results of the inhibition experiments it can by assumed that other isoenzymes of P-450 in addition to P450 IIEI may be involved in the conversion of Diglyme to 2-Methoxyethanol.
Executive summary:

Due to the high structural similarity of Triethylene glycol dimethyl ether and Diethylene glycol dimethyl ether (difference: one ethyl group; but same functional groups) it is very likely that both compounds will be metabolised by the same enzymes/metabolic path. Therefore, a read across from Triethylene glycol dimethyl ether to the metabolism data generated with Diethylene glycol dimethyl ether (Diglyme) is jusitfied to clarify the toxicokinetic behaviour of this substance (for further clarification please refer to the attached document "Proposed metabolism of Triethylene glycol dimethyl ether.doc").

Human and rat hepatic microsomes were incubated with Diethylene glycol dimethyl ether (Diglyme). The rat microsomes catalysed the NADPH-dependent cleavage of the central ether linkage of Diglyme yielding 2 -Methoxyethanol and 2 -(2 -Methoxyethoxy)ethanol. Microsomes isolated from phenobarbital- or ethanol-pretreated rats exhibited an increased capacity to cleave diglyme to 2 -Methoxyethanol. This ethanol-induced increase in 2 -Methoxyethanol formation was not observed if incubations contained the cytochrome P450 IIEI inhibitor Isoniazid. Pretreatment of rats with Diglyme significantly increased microsomal P-450 levels, P-450 associated enzyme activities and the conversion of Diglyme to 2 -Methoxyethanol.

Human hepatic microsomes also catalysed the NADPH-dependent cleavage of Diglyme to 2 -Methoxyethanol. The formation of 2 -Methoxyethanol from Diglyme correlated with the aniline hydroxylase activity (P450 IIEI) levels measured in human hepatic microsomes.

These results suggest that the ether linkages of Diglyme (and Triethylene glycol dimethyl ether) are cleaved by rat and human P-450 and 2 -Methoxyethanol is formed.