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Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The studies were performed to define formate-induced retinal dysfunction and histopathology in a rat modell of methanol intoxication.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
not applicable
Species:
rat
Strain:
Long-Evans
Sex:
male
Route of administration:
intraperitoneal
Vehicle:
other: saline
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
an initial dose of 4000 mg/kg followed by a supplemental dose of 1000 or 2000 mg/kg 12 h later
Remarks:
Doses / Concentrations:
4000 mg/kg initial dose
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 or 2000 mg/kg 12 h later
Basis:
nominal conc.
No. of animals per sex per dose:
not specified
Control animals:
yes
Details on results:
Methanol intoxicated rats developed formic acidemia, metabolic acidosis and visual toxicity within 36 hours.
Histopathological effect on retinal structure: In the high-dose group (7 - 15 mM blood formate vs. methanol-treated control with 0.5 to 2 mM formate), prominent vacuolation in the photoreceptors near the junction of inner and outer segments, with accumulation of densely stained material in the inner segments near the outer limiting membrane. Mitochondrial swelling and disruption was noted in the retinal pigment epithelium, photoreceptor inner segments and optic nerve (Eells et al., 2000; Seme et al., 2001).
Ultrastructural studies by electronmicroscopy revealed that the retinal morphology (as represented by the mitochdrial-rich, inner segment of the photoreceptor) was similar to the control after recovery of 72 h, but subtle photoreceptor changes were still present as a spacing between the cell nuclei of the outer nuclear layer which suggests residual histological alterations from formate-induced, previous edema (Seme et al., 2001). In the low-dose group (4 - 6 mM formate in blood), no histopathological changes were apparent at the light-microscopic level (Wallace et al., 1997). However, visual dysfunction was already visible in functional diagnostics, occurring as reductions in the flash evoked cortical potential (FEP) and in electroretinogram (ERG) at formate concentrations lower than those associated with morphological changes and provide functional evidence of direct retinal toxicity in methanol poisoning (Wallace et al, 1997). Rod- and cone-mediated ERG responses were attenuated in a formate- and time-dependent manner (Seme et al., 1999, 2001).
Biochemical effects: Retinal ATP, ADP, and GSH were significantly depleted following methonol-treatment under inhibition of formate oxidation after 72 and 144 h with GSH levels about 1/2 of controls, and after recovery still decreased, while energy metabolites showed no difference from the control values (Seme et al., 2001).

Functional tests provide functional evidence of direct retinal toxicity in methanol poisoning at stages not yet pronounced in histopathological changes. Hypothetical mechanism: Formic acid binds to cytochrome aa3 and inhibits cytochrome oxidase activity with inhibition constant values between 5 - 30 mM, which is in the range of concentrations found in the retina and vitreous humor of methanol-intoxicated rats. This may explain the effect on mitochondria and resulting visual dysfunction (Eells et al., 2000). The rat model appears to be a useful tool to also elucidate the sequelae of methanol intoxication in humans who are more sensitive than rodents.

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Principles of method if other than guideline:
Test model in monkeys for methanol-induced ocular toxicity after short-term exposure to characterize the toxicity syndrome and histological manifestations.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: oral
Species:
monkey
Strain:
other: Macaca mulatta
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.6-4.4 kg
Route of administration:
other: nasogastric tube
Vehicle:
water
Details on exposure:
VEHICLE
- Concentration in vehicle: 20 % (w/v)
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
approx. 1.5 to 6 days
Frequency of treatment:
variable
Post exposure period:
no data
Remarks:
Doses / Concentrations:
initially 2000 mg/kg, thereafter 500 mg/kg at variable frequencies and time points (exception: one animal 1000 mg/kg at 44 and 72 h and 2000 mg/kg at 144h)
Basis:
nominal conc.
No. of animals per sex per dose:
6 males in total
Control animals:
other: internal/same animal prior to treatment
Details on study design:
A high initial dose (2000 mg/kg) was followed by lower doses depending on the animal´s acidotic response in blood. Experience had told (McMartin et al., 1975) that after a single dose of 3000 mg/kg bw, in general, the animals died within 20 to 30 h without demonstrating ocular abnormalities. (Martin-Amat et al., 1977).
Examinations:
CAGE SIDE OBSERVATIONS: Yes (McMartin 1975, 3000 mg/kg)
- Time schedule: Continuously from application until death (approx. 33 h after application
- Cage side observations included: clinical signs, mortality

DETAILED CLINICAL OBSERVATIONS: Yes: cerebrospinal fluid pressure in cerebellomedullary cistern by cisternal puncture (2 animals)
- Time schedule: not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: one week before and during the course of intoxication, not further specified
- Dose groups that were examined: all animals: stereoscopic color fundus photography, fluorescein fundus angiography, pupillary light reflex,

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to treatment and throughout the course of the study
- Animals fasted: No data
- How many animals: all
- Parameters examined: blood pH, bicarbonate levels, methyl alcohol, blood formate (cerebrospinal fluid: 2 animals), pO2, pCO2

URINALYSIS: No

GROSS PATHOLOGY: No

HISTOPATHOLOGY: Yes (light and electron microscopic studies of neuronal tissues and nerve fibres associated with the eyes: retina, optic nerve heads, optic nerves of 2 control and 3 treated animals)
Positive control:
not applicable
Details on results:
OPHTHALMOSCOPIC EXAMINATION
The only detectable ocular change was optic disc edema (of the optic papilla). The primary sites of ocular injury were the optic nerve heads and the anterior segment of the optic nerve rather than the retinal ganglion cells themselves. In all eyes with optic disc changes, pupils were dilated and reacted poorly to light.

CLINICAL CHEMISTRY
Under methanol treatment acc. to this test design, formate levels were between min. 7.2 and max. 14.4 mEq/L in blood and 7.9 to 13.9 mEq/L in cerebrospinal fluid, blood bicarbonate min. 4.0 and max. 10.2 mEq/L, and blood pH min. 7.13 and max. 7.28. Methanol levels ranged from 1540 to 2840 mg/L (Martin-Amat et al., 1977).

HISTOPATHOLOGY: NON-NEOPLASTIC
All six animals developed fundus changes at the head of the optic nerve (optic disc) within 43 to 171 h after methanol ingestion, expressed as intraaxonal swellings (Hayreh et al, 1977). Electronmicroscopic studies revealed swelling of the nerve fibers with an accumulation/clustering of swollen mitochondria in the optic nerve head being maximally in the lamina cribrosa region. Furthermore, in the retrolaminar and intraorbital optic nerve, swelling of astrocytes was prominent as well as swelling of the cytoplasm of the oligodendroglial cytoplasm in contact with the axons (Baumbach et al., 1977). Alterations were not observed in the retina itself: the ganglion cells of the retina were intact with only minimal swellings of the mitochondria and loss of cristae. But these findings were also present in the control tissue (Baumbach et al., 1977).

While acute methanol toxicity in monkeys (after a single dose) does not yield ocular signs, repeated dosing succeeded in producing ocular lesions (Martin-Amat et al, 1977).

The only detectable ocular change was optic disc edema (of the optic papilla) which was similar to that seen in raised intracranial pressure in humans, but without this pressure after methanol (Hayreh et al, 1977). The primary sites of ocular injury were the optic nerve heads and the anterior segment of the optic nerve rather than the retinal ganglion cells themselves. It appears that interference with oxidative phosphorylation causes mitochondrial damage, thus disruption of active axoplasmic flow in the retrolaminar optic nerve (Baumbach et al., 1977; Hayreh et al., 1977). [note: In humans it has been hypothesized that optic atrophy, which often follows acute methanol intoxication, is secondary to injury of the retinal ganglion cells.]. Mechanistically, there is a close causal relationship between the prolonged increase in formic acid from methanol and the development of optic edema. Similar effects can be produced by intravenous administration of formate without acidosis (Martin-Amat et al., 1978).

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The study was designed to determine whether components of folate-dependent formate oxidation, (folate and 10-formyltetrahydrofolate dehydrogenase (10-FDH)) exist in retina and whether differences in these components might explain species-determined susceptibility to methanol intoxication. No methanol was administered.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
not applicable
Species:
other: rat and human
Strain:
other: rat: Sprague-Dawley
Sex:
not specified
Route of administration:
other: no administration
Vehicle:
other: no administration
Details on results:
The cell-specific localisation of the enzyme, 10-FDH, was found to be similar in rat and human retina, preferentially located in the Müller-cell type, the principal glia of the retina (by immunohistochemistry).
The amount of 10-FDH found in cytosolic as well as in the mitochondrial fraction, was about 3x higher in humans than in rats (Western blot analysis). However, the retinal folate levels were lower in humans (about 14 % of that in rats), compared with the high folate liver pools, the retina contains very much less folate.
Endpoint:
behavioural effects
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The study was designed to examine neurobehavioral toxicity in methanol-induced rats.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
neurotoxicity
Species:
rat
Strain:
Long-Evans
Sex:
male
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
single dose
Frequency of treatment:
only one dose
Post exposure period:
10 min
Remarks:
Doses / Concentrations:
1000, 2000, 3000 mg/kg (50-% aqueous solution)
Basis:
nominal conc.
No. of animals per sex per dose:
11 (The same rats served as their own controls and were re-used for each dose on different days.)
Control animals:
other: test animal = control group (water) prior to treatment
Details on results:
The rats displayed no signs of overt intoxication such as gait disturbance, but a significant, dose-related reduction in FR20 response was observed at all dose-levels.
A NOAEL for behavioural changes cannot be derived.

Description of key information

LOAEL (ocular toxicity) intraperitoneal = 5000 mg/kg bw for rats (no NOAEL identified)

Additional information

In a study by Eells et al. (2000), rats were intraperitoneally dosed with methanol. In all of these animals, the folate-dependent formate-oxidation was inhibited. After the initial dosage of 4000 mg/kg bw, 12 hours later an injection of 1000 or 2000 mg/kg bw followed. Formic acidemia, metabolic acidosis and visual toxicity occurred (Eells et al., 2000). Histopathology demonstrated vacuolation in the photoreceptors, mitochondrial swelling and mitochondrial disruption in the retinal pigment epithelium, which were dependent on blood formate levels. However, functional changes could already be demonstrated by electroretinogram (ERG) and flash evoked cortical potential (FEP) in animals not showing morphological changes, 72 hours of recovery. These functional tests provide functional evidence of retinal toxicity in methanol poisoning at stages not yet pronounced as histopathological changes. The authors stated the hypothetical mechanism that formic acid binds to cytochrome aa3 and inhibits cytochrome oxidase activity with inhibition constants between 5 and 30 mM, which is in the range of concentrations found in the retina and vitreous humour of methanol-administered rats. This may explain the effect on mitochondria and resulting visual dysfunction (Eells et al., 2000).

Formate oxidation was found to be about 50% lower in human than in rat retina (Eells et al., 1995). This is in line with the finding that lower folate levels in human retina may limit the conversion of formate into CO2 and contribute to the high ocular toxicity in humans.

A subacute oral toxicity study in monkeys indicated that repeated methanol dosing caused ocular lesions after a high initial dose of 2000 mg/kg bw followed by lower doses for up to 6 days, depending on the animal´s acidotic response in blood (Martin-Amat et al., 1977), while acute methanol toxicity did not yield signs of ocular toxicity (McMartin et al., 1975). The only detectable ocular change was optic disc edema (of the optic papilla) which was similar to that seen in raised intracranial pressure in humans, but without this pressure after methanol (Hayreh et al., 1977). The primary sites of ocular injury were the optic nerve heads and the anterior segment of the optic nerve rather than the retinal ganglion cells themselves. It appears that interference with oxidative phosphorylation causes mitochondrial damage; thus, disruption of active axoplasmic flow in the retrolaminar optic nerve (Baumbach et al., 1977; Hayreh et al., 1977). Mechanistically, there is a close causal relationship between the prolonged increase in formic acid resulting from methanol and the development of optic edema. Similar effects can be produced by intravenous administration of formate without acidosis (Martin-Amat et al., 1978).