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Description of key information

Oral: LOAEL subacute = 2340 mg/kg/bw in monkeys (mortality 7/7 after 3 d exposure) 
Inhalation: NOAEC chronic = 0.013 mg/L air in monkeys (7 to 29 months exposure)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
other: not specified
Principles of method if other than guideline:
Daily application of a single dose of methanol to monkeys by gavage over a period of 3 days.
GLP compliance:
not specified
Limit test:
no
Species:
monkey
Strain:
not specified
Sex:
male
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 % solution
- Amount of vehicle (if gavage): no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 days
Frequency of treatment:
daily
Dose / conc.:
2 340 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
7 males
Control animals:
not specified
Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
2340 mg/kg bw was the lethal dose for all 7 animals after 3 days.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
LOAEL
Effect level:
2 340 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
mortality
Key result
Critical effects observed:
no

2340 mg/kg bw was the lethal dose for all 7 animals under test after 3 days.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
2 340 mg/kg bw/day
Study duration:
subacute
Species:
monkey

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
limited documentation
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Comprehensive study programme on monkeys including metabolic, pharmacokinetic and short-, long-term studies, reproductive assays and carcinogenicity studies.
GLP compliance:
not specified
Limit test:
no
Species:
monkey
Strain:
Macaca fascicularis
Sex:
not specified
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: 4 monkeys were housed in one inhalation chamber.
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical values close to nominal ones.
9.9 ± 1.3 ppm
101.0 ± 8.2 ppm
1015.6 ± 82.7 ppm
Duration of treatment / exposure:
a) 7 months
b) 1 year + 7 months (19 months)
c) 2 years + 5 months (29 months)
Frequency of treatment:
21 h/d
Dose / conc.:
0.013 mg/L air (nominal)
Remarks:
corresponding to 10 ppm
Dose / conc.:
0.13 mg/L air (nominal)
Remarks:
corresponding to 100 ppm
Dose / conc.:
1.3 mg/L air (nominal)
Remarks:
corresponding to 1000 ppm
No. of animals per sex per dose:
a) 7 months: 2 monkeys
b) 1 year + 7 months (19 months): 3 monkeys
c) 2 years + 5 months (29 months): 3 monkeys
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Clinical observations and clinical-chemical, hematological and ophthalmological examinations were done.
Sacrifice and pathology:
Sacrifices were conducted at 7 months (2 monkeys), at 19 months (3 monkeys), and at 29 months (3 monkeys) (Tab. 3, p. 29).

GROSS PATHOLOGY and HISTOPATHOLOGY
brain, heart, liver, lung, trachea, kidney, peripheral nervous system
Other examinations:
Clinical, clinical-chemical, hematological and ophthalmological were done.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The monkeys of the 1.3 mg/L group showed abnormal scratching of the skin and clinical symptoms such as frequent yawning and nasal discharge (see also recovery test, p. 31 f).
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The animals showed normal body-weight gain, food and and water consumption throughout.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Examinations of the eye fundus revealed no changes in any group.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological examinations revealed no deviations from normal in Hb, Ht, red and white blood-cell count, or percentage of white blood cells in any group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant differences in serum values were seen. However, one monkey in the 0.13 mg/L group showed differences for most liver parameters (total protein, GOT, GPT, total cholesterol, free cholesterol, thymol and glucose). This was not considered treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
BRAIN:
In both upper exposure groups, the number of responsive stellate cells containing increased endoplasmic reticulum (minimal hypertrophy of astroglia) appeared in the basal ganglions after 1 year and 7 months of exposure. In the cerebral white substance, slight hyperplasia of the astroglia was noted in the 0.13 and 1.3 mg/L groups, in particular in the 0.13 mg/L group after 7 months (NEDO, 1987, p.53). Yet, this was not characteristic of a degenerative process and was unrelated to dosing, because no such phenomena were noted after 2 years and 5 months.
In the cerebral white substance, diffuse increases of responsive stellate cells centered in the subcortical white substance and the semioval center of the frontal and parietal lobes was noted at the higher dose groups, but also emerged in 3/8 monkeys of the 0.013 mg/L group after 29 months (p. 23). This was apparently a reversible effect, as shown from monkeys after recovery.
There were a few cases of degeneration of the optic nerve and the corpus geniculatum laterale after 7 months and 19 months (ophthalmencephalon). In one or two cases in the groups of 8 animals exposed for 2 years and 5 months, including the 0.013 mg/L group, slight degeneration of the optic nerve was suspected, but not considered as significant (p. 23). Slight degeneration processes including increased responsiveness of the astroglia were noted in the thalamus after exposure to 0.13 and 1.3 mg/L for 7 months and longer (p. 24).

LUNG and TRACHEA:
There were no cases of pneumonia in any of the exposed monkeys. In the lungs, fibrosis was seen in the interalveolar space. However, no dose response was observed, and the effect was also seen in the controls. In the trachea, atrophy of the epithelium of the mucous membranes and reduction of goblet cells were observed in a total of 4 cases of exposed animals, but not correlated with the amount of methanol inhaled. This was not seen in the controls (p. 28).

Peripheral Nervous System:
One monkey at 0.13 mg/L and 2 at 1.3 mg/L showed slight but clear changes in peroneal nerves; the authors concluded that these effects show that methanol causes damage to peripheral nerves (p. 25/26).

Liver and Kidney:
There were mild degenerations of the livers and kidneys which showed no clear correlation with exposure levels and exposure time. There was some evidence of an increase in fatty granules in liver parenchyma at 1.3 mg/L along with signs of fibrosis in the hepatic cord more pronounced than at the lower concentration. Sudan-positive granules were observed at 0.13 and 1.3 mg/L in the renal tubular epithelium (NEDO, 1987, p. 26/27).

Heart:
There was an increase in the Sudan-positive granules in heart tissue at 1.3 mg/L, but not manifested in any morphological lesion of the heart muscle (e.g. fibrosis).
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In both upper dose groups, the ECG of one of the 0.13 mg/L exposed monkeys and three of the 1.3 mg/L exposed monkeys showed abnormalities (negative T- or Q-wave changes and flattening of T-wave) which indicated slight myocardial disorder (p. 21).
Details on results:
CLINICAL SIGNS AND MORTALITY
The monkeys of the 1.3 mg/L group showed abnormal scratching of the skin and clinical symptoms such as frequent yawning and nasal discharge (see also recovery test, p. 31 f).


BODY WEIGHT GAIN AND FOOD AND WATER CONSUMPTION
The animals showed normal body-weight gain, food and and water consumption throughout.


OPHTHALMOSCOPIC EXAMINATION
Examinations of the eye fundus revealed no changes in any group.


HAEMATOLOGY
Haematological examinations revealed no deviations from normal in Hb, Ht, red and white blood-cell count, or percentage of white blood cells in anygroup.


CLINICAL CHEMISTRY
No significant differences in serum values were seen. However, one monkey in the 0.13 mg/L group showed differences for most liver parameters (total protein, GOT, GPT, total cholesterol, free cholesterol, thymol and glucose). This was not considered treatment-related.


HISTOPATHOLOGY

BRAIN:
In both upper exposure groups, the number of responsive stellate cells containing increased endoplasmic reticulum (minimal hypertrophy of astroglia) appeared in the basal ganglions after 1 year and 7 months of exposure. In the cerebral white substance, slight hyperplasia of the astroglia was noted in the 0.13 and 1.3 mg/L groups, in particular in the 0.13 mg/L group after 7 months (NEDO, 1987, p.53). Yet, this was not characteristic of a degenerative process and was unrelated to dosing, because no such phenomena were noted after 2 years and 5 months.
In the cerebral white substance, diffuse increases of responsive stellate cells centered in the subcortical white substance and the semioval center of the frontal and parietal lobes was noted at the higher dose groups, but also emerged in 3/8 monkeys of the 0.013 mg/L group after 29 months (p. 23). This was apparently a reversible effect, as shown from monkeys after recovery.
There were a few cases of degeneration of the optic nerve and the corpus geniculatum laterale after 7 months and 19 months (ophthalmencephalon). In one or two cases in the groups of 8 animals exposed for 2 years and 5 months, including the 0.013 mg/L group, slight degeneration of the optic nerve was suspected, but not considered as significant (p. 23). Slight degeneration processes including increased responsiveness of the astroglia were noted in the thalamus after exposure to 0.13 and 1.3 mg/L for 7 months and longer (p. 24).


LUNG and TRACHEA:
There were no cases of pneumonia in any of the exposed monkeys. In the lungs, fibrosis was seen in the interalveolar space. However, no dose response was observed, and the effect was also seen in the controls. In the trachea, atrophy of the epithelium of the mucous membranes and reduction of goblet cells were observed in a total of 4 cases of exposed animals, but not correlated with the amount of methanol inhaled. This was not seen in the controls (p. 28).


Peripheral Nervous System:
One monkey at 0.13 mg/L and 2 at 1.3 mg/L showed slight but clear changes in peroneal nerves; the authors concluded that these effects show that methanol causes damage to peripheral nerves (p. 25/26).

Liver and Kidney:
There were mild degenerations of the livers and kidneys which showed no clear correlation with exposure levels and exposure time. There was some evidence of an increase in fatty granules in liver parenchyma at 1.3 mg/L along with signs of fibrosis in the hepatic cord more pronounced than at the lower concentration. Sudan-positive granules were observed at 0.13 and 1.3 mg/L in the renal tubular epithelium (NEDO, 1987, p. 26/27).

Heart:
There was an increase in the Sudan-positive granules in heart tissue at 1.3 mg/L, but not manifested in any morphological lesion of the heart muscle (e.g. fibrosis).


OTHER FINDINGS
In both upper dose groups, the ECG of one of the 0.13 mg/L exposed monkeys and three of the 1.3 mg/L exposed monkeys showed abnormalities (negative T- or Q-wave changes and flattening of T-wave) which indicated slight myocardial disorder (p. 21).


Key result
Dose descriptor:
NOAEC
Effect level:
0.013 mg/L air (nominal)
Sex:
not specified
Basis for effect level:
other: slight myocardial effects and slight hyperplasia of the astroglia in the cerebral white substance
Key result
Dose descriptor:
LOAEC
Effect level:
0.13 mg/L air (nominal)
Sex:
not specified
Basis for effect level:
other: slight myocardial effects and slight hyperplasia of the astroglia in the cerebral white substance
Key result
Critical effects observed:
no

Taking into account slight myocardial effects and slight hyperplasia of the astroglia in the cerebral white substance

already noted at 0.13 mg/L, then already 0.13 mg/L have to be defined as LOAEL with a NOAEL of 0.013 mg/L.

But no clear adverse effects are reported at 1.3 mg/L. No necrotic effects occurred in the nervoustissue. Hyperplasia of astroglia, not considered as degenerative, might be a transient methanol-dependent effect which appears to subside upon cessation of long-term

methanol exposure (p. 52/53). But there was no clear correlation to the exposure concentration and time. Therefore, the biological relevance is questionable. There was evidence of an increase in mild fatty changes in the liver and kidney upon exposure to 1.3 mg/L, which were associated with signs of fibrosis. This effect was still present after recovery (see other entry). Given the small histological effect (p. 27), the pathological relevance appears to be low, but indicates that long-term exposure to 1.3 mg/L methanol is on the borderline to toxicologically relevant, histological manifestations (authors statement, p.27).

There were histological signs of diffuse responsiveness of the astroglia in some parts of the cerebral white substance at all exposure levels, at high exposure after shorter time period, after 0.013 mg/L after 29 months in a few animals. The relevance was not clearly commented by the authors. However, under test conditions (continuous exposure), there was no evidence of irreversible effects arising from long-term exposure to up to 1.3 mg/L methanol. Therefore, the NOAEL for continuous exposure may be established at 1.3 mg/L, but the low number of animals and limited description do not allow to draw firm conclusions on the apparent neural effect.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
13 mg/m³
Study duration:
chronic
Species:
monkey

Additional information

Animal data

Oral:

Seven male monkeys received daily doses of 2340 mg/kg bw methanol as 30% aqueous solution by oral gavage for three days. Under the test conditions, this dosage was lethal for all seven animals (Rao et al., 1977).

Inhalation:

In a whole-body inhalation study in monkeys exposed to 0.013, 0.13, and 1.3 mg/L for 21 hours/day, 7 days/week for 7, 19, and 29 months, several general clinical signs as well as degenerative effects in the brain (at 0.13 and 1.3 mg/L), slight peripheral nerve damage (at 0.13 and 1.3 mg/L), very slight degeneration of the optic nerve (concentrations not noted), increased fat granules and slight fibrosis in the liver (all concentrations) as well as Sudan positive granules in the kidney were observed (at 0.13 and 1.3 mg/L). Also, a slight myocardial disorder (at 0.13 and 1.3 mg/L) and localized effects in the trachea and possible slight fibrosis in the lungs (concentrations not noted) were observed. Although the statistical significance of the effects cannot be verified from the limited study report, the effects observed appear to be associated with methanol (NEDO, 1987).

In a short-time experiment, monkeys were exposed up to 20 days for 21 hours per day to methanol vapour. Coma and lethality were observed at concentrations > 9.31 mg/(L*d). In the brain, necrosis of the basal ganglia and cerebral edema were observed at 6.65 mg/(L*d) and at 3.99 mg/(L*d), hyperplasia and fibrosis around myelin sheaths of the basal ganglia as well as a slight to moderate increase in astroglia cells were observed. The optic nerve showed atrophy at > 3.99 mg/(L*d), along with reduction in myelin fibers. In the liver, fibrosis was observed at 6.65 mg/(L*d) and mild fatty degeneration was observed at 3.99 mg/(L*d). In the kidney, partly vacuolated hyaline degeneration was observed at 6.65 mg/(L*d) (NEDO, 1987). The liver and kidney effects were recorded at doses already overtly toxic in humans and, hence, are of low relevance.

In rats exposed to methanol up to 6.65 mg/L for 6 hours per day, five days per week for 28 days, no adverse effects were observed except local nasal irritation and increased relative spleen weights, which were observed only at the middle dose. The estimated blood level of methanol was about 250 mg/L under this condition (Andrews et al., 1987).

In a whole-body inhalation study in mice exposed for 12 months to concentrations of 0.013, 0.13, and 1.3 mg/L for 20 hours/day, slight changes in clinical signs, body and organ weights, and some changes in histopathology were observed, but these effects were considered to be toxicologically irrelevant (NEDO, 1987). In rats exposed in the same manner, slight changes in body weight and organ weights were observed at the highest dose. The NOEC was 0.13 mg/L, the NOAEC was 1.3 mg/L for rats and mice in these studies (NEDO, 1987). Again, these effects are of low relevance in the light of the onset of human toxicity already at lower doses. The species-related differences are very obvious between rodents and primates.

The latter demonstrating a 100-fold greater susceptibility for methanol-related effects due to differences in metabolism of methanol. In rodents methanol is metabolized to carbon dioxide to a great extent, whereas in primates formate accumulation is responsible for the observed effects.

Human data:

In male and female workers exposed to methanol from 0.3 to 7.8 years, the highly exposed workers (4.7 - 7.3 mg/L) more often complained of blurred vision, headache and nasal irritation during or after work. Nobody stated to suffer from photophobia. The examination of the eye fundus failed to reveal retinal changes. Among three workers exposed to about 1.0 to 1.6 mg/L and one worker exposed to 0.12 to 3.6 mg/L, two showed retarded pupil reflex and one exhibited mild mydriasis (Kawai et al., 1991). Other common complaints were forgetfulness and skin sensitivity (IPCS/WHO, 1997).

A health hazard evaluation was conducted by the National Institute for Occupational Safety and Health (NIOSH) to determine if vapours from duplicating fluid (99% methyl alcohol) used in direct-process spirit duplicating machines were causing adverse health effects among teacher aides (Frederick et al., 1984). The teacher aides reported significantly more blurred vision, headache, dizziness, and nausea than the comparison group. Concentrations of airborne methyl alcohol ranged from 0.48 to 4.0 mg/L. Additional studies also showed that headaches were associated with occupations that involve the operation of duplicating machines (NTP, 2003; IPCS/WHO, 1997).


Repeated dose toxicity: via oral route - systemic effects (target organ) neurologic: eyes (retina, optic nerve)

Repeated dose toxicity: inhalation - systemic effects (target organ) cardiovascular / hematological: heart; digestive: liver; neurologic: brain (multiple sections)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The experimental studies with animals do not provide clear evidence for the necessity for classification: In primates a potential of methanol to cause adverse health effects was shown, while in rodents only toxicologically irrelevant effects were shown. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.