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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1983

Materials and methods

Objective of study:
distribution
metabolism
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The in vivo and in vitro metabolism of Morpholine was assessed through the investigation of the subcellular binding interactions, the generation of nitrosamines during Morpholine metabolism, biologically active Morpholine metabolites in a UDS assay, the biochemical basis for species differences noted in Morpholine metabolism, and the differences in human, rat, hamster and guinea pig liver capacity for Morpholine metabolism.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Morpholine
EC Number:
203-815-1
EC Name:
Morpholine
Cas Number:
110-91-8
Molecular formula:
C4H9NO
IUPAC Name:
morpholine
Test material form:
liquid
Radiolabelling:
yes

Test animals

Species:
other: Sprague Dawley rats, strain II guinea pigs, human liver biopsy samples
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
Please refer to "Any other information on materials and methods incl. tables"

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
not specified
Details on exposure:
Please refer to "Any other information on materials and methods incl. tables"
Duration and frequency of treatment / exposure:
Study 1: single injection
Study 2: single injection
Study 3: single administration
Study 4: not applicable (analytical study)
Study 5: single injection
Study 6: not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
Study 1: 125 mg/kg bw (50 or 100 µCi/animal)
Study 2: 200 µCi/animal
Study 3: 50 or 200 µCi/animal
Study 4: not applicable (analytical study)
Study 5: no data
Study 6: not applicable
No. of animals per sex per dose / concentration:
no data
Control animals:
yes
Positive control reference chemical:
no
Details on study design:
No rationale for dose level selection was provided.
Details on dosing and sampling:
Please refer to "Any other information on materials and methods incl. tables"
Statistics:
no data

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
1. Uniform distribution of 14C in TCA-insoluble fractions indicated a non-specific binding and/or incorporation of Morpholine. No significant amounts of covalently bound 14C were detected in the subcellular fractions of the liver.

2. Very little, if any, 14C was bound to liver DNA.

Any other information on results incl. tables

In this report including in vivo and in vitro metabolism studies, the metabolism of Morpholine was assessed through the investigation of subcellular binding interactions, nitrosamines generation as part of Morpholine metabolism, biologically active Morpholine metabolites in a UDS assay, the biochemical basis for species differences noted in Morpholine metablism, and the differences in human, rat, hamster and guinea pig liver capacity for Morpholine metabolism.

A uniform distribution of 14C in TCA-insoluble fractions indicated a non-specific binding and/or incorporation of Morpholine. No significant amounts of covalently bound 14C were detected in the subcellular fractions of the liver. Very little, if any, radioactivity derived from 14C Morpholine was bound to liver DNA. Therefore, it was concluded that the TCA-insoluble macromolecules of the nuclear fraction probably reflected the 14C associated with proteins and/or RNA. The results did not provide evidence for the in vivo formation of N-nitrosomorpholine after administration of Morpholine under the conditions described. In vivo methylation of Morpholine (step 1 in the metabolism process) proceeded via the SAM pathway. Guinea pig liver microsomes showed the most metabolic activity in vitro. Ziegler's flavin monooxygenase was exlusively involved in the N-oxidation of N-methylmorpholine and at least partially so in the N-hydroxylation of Morpholine and was evidence against the involvement of cytochrome P450 in these reactions. Of the species examined, human liver most resembled that of the rat in its ability to metabolize Morpholine.Morpholine was not bound to serum proteins.

This metabolism study is classified as acceptable.

1. In the initial part of this study, the amount of 14C in the tissue macromolecules, expressed as µg eq. of Morpholine/g tissue, remained rather constant in all organs throughout a 24 hour period. In contrast, the total radioactivity determined in the whole tissue homogenates (representing bound plus unbound forms) declined rapidly with time. Since a considerable amount of 14C was found to be present in fractions from the liver and kidney homogenates, a second phase was conducted to determine whether there was preferential uptake in some particular subcellular fraction. While cytosol contained the highest level of 14C (expressed in terms of ng eq. Morpholine/µg protein) in both the liver and kidney, when 14C was determined in various subcellular fractions no signficant differences among these were detected.

2. The specific activity of the DNA was 31 and 29 dpm per mg DNA for 4 and 24 hours, respectively. The U.V. absorption monitored at 260 nm clearly demonstrated the presence of guanine and adenine, eluted at 6 and 12 mL and pyrimidine oligonucleotides eluting between 2 and 5 mL, respectively. However, no significant radioactivity associated with either peak or at any other elution volume were detected.

3. In no case was any radioactivity above background found to be associated with N-nitrosomorpholine peak in either HPLC system.

4. Not applicable.

5. When N-methylmorpholine-N-oxide was isolated from the urine (in vivo), 10.5 % of the radioactivity of the L-[methyl-14C]-methionine administered was found to be incorporated in the metabolite. When tested in vitro, the initial rate of formation of N-methylmorpholine by guinea pig liver cytosol was approximately 0.13 nmol/min/mg protein, while the rate using hamster liver cytosol was less than 1/10 of this and was almost undetectable using rat liver cytosol. When evaluating the metabolic rates of oxidation of N-methylmorpholine to N-methylmorpholine-N-oxide or of Morpholine to N-hydroxymorpholine, the N-hydroxylase activity was almost undetectable using rat liver microsomes; however, hamster liver microsomes contained significant levels of this enzyme and guinea pig liver microsomes were even more active. When the liver microsome catalyzed N-oxidation of N-methylmorpholine was evaluated, liver microsomes from the guinea pig were more active than those from the hamster, which, in turn, were more active than rat liver microsomes. While the species differences in the rates of N-oxidation were not as marked as those observed in the case of N-methylation or N-hydroxylation of Morpholine, a similar pattern was observed, with liver microsomes from the guinea pig being more active than those from the hamster, which, in turn, were more active than rat liver microsomes. When in vitro incubations were conducted in the presence of methimazole, SKF 525 -A and liver microsomes, SKF 525 -A failed to inhibit N-hydroxylation of Morpholine and the N-oxidation of N-methylmorpholine; however, methimazole inhibited the N-hydroxylation by approximately 60 % and almost completely blocked the N-oxidation of methylmorpholine.

6. Using human liver cytosols, no detectable Morpholine N-methylase activity was found. When the formation of N-methylmorpholine-N-oxide from N-methylmorpholine was monitored using microsomes, human livers were found to be active in carrying out this reaction, though the activity was lower than that observed with rat liver. In addition, studies using enzyme modifiers showed that the reaction was inhibited 70% by methimazole, indicating that the flavin-containing monooxygenase was also involved in the human liver in carrying out this reaction. Determination of Morpholine hydroxylase indicated that the human liver microsomes were able to N-hydroxylate Morpholine, with an activity of approximately 0.008 µmol/mg protein/30 minutes.

Applicant's summary and conclusion