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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Toxicological Research of Methanol as a fuel for Power Station. Summary Report on Tests with Monkeys, Rats and Mice.
Author:
New Energy Development Organization
Year:
1987
Bibliographic source:
New Energy Development Organization, Tokyo
Reference Type:
publication
Title:
Proposal for reference concentrations (RfC) for inhalation exposure to methanol.
Author:
Vyskocil, A. and Viau, C.
Year:
2000
Bibliographic source:
Environ. Toxicol. Pharmacol. 9, 9-18

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
monkey
Strain:
Macaca fascicularis
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: 4 monkeys were housed in one inhalation chamber.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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).


Effect levels

open allclose all
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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion