Methylhydrazine

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

hematoxicity

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, non-guideline, mechanistic study of relevance for risk assessment of hemolytic effects.

Qualifier:

no guideline required

Principles of method if other than guideline:

mechanistic study

GLP compliance:

no

Remarks:

prior to GLP

Type of method:

in vitro

Species:

human

Details on test animals or test system and environmental conditions:

Venous blood was collected from normal human subjects in heparinized tubes, centrifuged and the plasma and buffy coat removed. The red
cells were washed three times with phosphate buffered isotonic saline, pH 7.4, containing 0.01M glucose and resuspended in the same buffer at a hematocrit of 40-50%.

Route of administration:

other: in vitro

Vehicle:

other: see above

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

incubated at 37°C, samples taken for the various determinations at two, four, six and eight-hour intervals.

Frequency of treatment:

single

Post exposure period:

none

Remarks:

Doses / Concentrations:
10, 1 and 0.1 mM
Basis:
nominal conc.

No. of animals per sex per dose:

NA

Control animals:

yes, concurrent vehicle

Examinations:

- total red cell counts, hematocrit and hemoglobin levels
- osmotic RBC fragility test (Dacie, 1954)
- percent methemoglobin (Hainline, 1965)
- reduced glutathione levels (Patterson, 1955)
- staining for incidence of cells with Heinz bodies (Methyl violet in 0.9% saline)
- glucose consumption, lactate production, ATP levels (on protein free filtrates)
- RBC potassium level
- Malonyldialdehyde (MDA) levels were measured as an indication of membrane lipid peroxidation.

Positive control:

none

Details on results:

- total red cell counts, hematocrit and hemoglobin levels: no data
- MDA levels, osmotic RBC fragility: no significant changes
- % methemoglobin: none at 0.1 mM x 6h, 10% and 22% at 1 and 10 mM x 2h, reversible
- GSH levels: significant decrease at 10 mM, peak drop at 4h, reversible
- % cells with Heinz bodies: none at 0.1 mM x 6h; 20% and >90% at 1 and 10 mM x 2h.
- glucose consumption, lactate production: significantly increased at 10 mM
- ATP levels: no significant effect (no apparent interference with the glycolytic process)
- RBC potassium level: decreased at 10 mM x 6h

Executive summary:

Human RBC were exposed to MMH for various durations (2-8h) in vitro. Effects were dose-dependent and generally observed quickly.

MMH, although a strong reducing agent produces effects in the red cells characteristic of oxidative damage. It has been postulated that MMH in the presence of oxygen forms methyldiazine which further reacts to provide a potent source of free radicals. The oxidation of intracellular constitutents indicated by the formation of methemoglobin and Heinz bodies and the oxidation of GSH supports this view.

The data show no severe irreversible effects on intracellular RBC metabolism from MMH exposure, except for Heinz body formation, leading to increased cell rigidity which probably causes an increased rate of cell sequestration and destruction in the spleen.

An apparent NOAEC, for only 6h exposure, was 0.1 mM of MMH in the test medium (which was not complete blood).

Additional information

MMH induces seizures in several mammal species, with various candidate antidotes which should be carefully investigated for their safety and efficiency by a physician before any use. However, use in SCC should already render such effects unlikely (no acute exposure).

MMH interacts with RBC structure and metabolism, leading notably to methemoglobin formation.