Sodium methanolate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

- limited documentation

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 6 weeks
- Housing: The animals were housed individually in wire-mash cages attached to the inhalation chamber.
- Diet (e.g. ad libitum): solid chow for mice (CRF-1, Charles River Japan Inc.)
- Water (e.g. ad libitum): filtered and sterilised tap water
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton 1000 Inhalation Exposure Chamber

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of chamber concentration: analytical values close to nominal ones.

Duration of treatment / exposure:

12 months (males: 7202-7225 h; females: 7352-7373 h)

Frequency of treatment:

continuously, mean daily exposure time: 19.8 hours

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:

30

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Measurements were carried out on all animals once a week.


FOOD CONSUMPTION:
- The measurements were done weekly during the first 13 weeks of exposure, and monthly thereafter.


HAEMATOLOGY: Yes


CLINICAL CHEMISTRY: Yes


URINALYSIS: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
For interim examination, 10 animals per sex and dose were sacrificed after 6 months.

Statistics:

All the data obtained were analysed by t-test, Fischer´s exact test or Armitage´s chi-square test as appropriate for any significant difference.

Clinical signs:

no effects observed

Description (incidence and severity):

During the whole study, there were no changes in the general appearance that could suggest any relation to methanol treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal of the 0.13 mg/L group died on day 174, another female animal (also of the 0.13 mg/L group) had to be sacrificed in moribund state on day 178.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body-weights of both sexes of the treated groups tended to be higher than the controls in the second half of the study (NEDO, Fig. 3, p. 168).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was decreased food consumption in high-dose females during the first 2 months and from month 7 throughout the study.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

All clinical and blood tests failed to reveal any methanol-related alteration in parameters from normal.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All clinical and blood tests failed to reveal any methanol-related alteration in parameters from normal.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinary protein was in the normal range of physiological variations. None of pH, glucose, ketones, bilirubin, occult blood or urobilinogen showed any changes suggesting effects from the treatment.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the group of high-dose females, an increase in organ weight with significant differences was noted for the liver after 6 months and for the kidney and spleen after 12 months, however, the latter without statistical significance and a clear dose-response.
The organ/body weight ratio did not show any significant difference.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed various non-tumoral changes. Except for vacuolar degeneration of the ureter epithelium at the kidney, all findings were observed due to aging or of accidental nature commonly seen in mice of this age and strain (6 or 12 month old, B6C3F1).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy of animals sacrificed after 6 or 12 months of treatment revealed a sporadical incidence of accidental changes for both males and females. But there were no changes which occurred in a high incidence or specifically in the treated groups.
The two females of the mid-dose group (0.13 mg/L) which died or were sacrificed in extremis had thoracic or abdominal dropsy, atrophy of the thymus and spleen, severe roughness and grey-coloration of the kidney surface.
Liver: After 12 months, the incidence of more severe fatty degeneration of hepatocytes was pronounced in high-dose males: This was graded as moderate in 8/20, mild in 8/20 and negative in 4/20 surviving males vs. 1/20 (moderate), 9/20 (mild) and 10/20 negative in the male control group. On the other hand, this effect was common and found to similar extent in untreated/external male mice. The female mice showed no difference among the groups compared to control.

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: histopathological examinations; body weight; food consumption; organ weights

Key result

Dose descriptor:

NOEC

Effect level:

0.13 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: histopathological examinations; body weight; food consumption; organ weights

Key result

Critical effects observed:

no

Conclusions:

According to the authors, only in the 1.3 mg/L dose-group changes could be considered treatment-related. But based on the minor degree and severity, these changes have no pathological meaning and are considered as toxicologically irrelevant. Levels of 0.13 mg/L or less did not produce any effect (=NOEC). 1.3 mg/L can be established as NOAEC, while it is the LOEC for non-pathological, apparently treatment-related effects.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

- limited documentation

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc.
- Housing: The animals were housed individually in wire-mash cages attached to the inhalation chamber.
- Diet (e.g. ad libitum): solid chow for rats (CRF-1, Charles River Japan Inc.)
- Water (e.g. ad libitum): filtered and sterilised tap water
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

not specified

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton 1000 Inhalation Exposure Chamber

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of chamber concentration: analytical values close to nominal ones.

Duration of treatment / exposure:

12 months (total exposure time: 7318-7341 h: males; 7474 - 7496 h: females)

Frequency of treatment:

continuously, average about 20 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 100ppm

Dose / conc.:

1.3 mg/L air (nominal)

Remarks:

corresponding to 1000 ppm

No. of animals per sex per dose:

20

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes


FOOD CONSUMPTION:
- The feed to be consumed during a week was determined for two animals, and the daily food consumption per animal was then calculated. The calculation was done weekly during the first 13 weeks of exposure, and monthly thereafter.


HAEMATOLOGY: Yes


CLINICAL CHEMISTRY: Yes


URINALYSIS: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

All the data obtained were analysed by t-test, Fischer´s exact test or Armitage´s chi-square test as appropriate for any significant difference.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat of the 1.3 mg/L dose group and one male rat of the 0.013 mg/L dose group died or were sacrificed in extremis (on day 337 and on day 340, respectively). This was considered spontaneous on the basis of the absence of a consistent dose relationship.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A very slight transient suppression of body-weight gain was observed for males and females of the 1.3 mg/L dose group from week 27 through 44 (less than 5 %). This has to be attributed to temporary occurrence of slight diarrhea in these groups during this period. The otherwise addressed significant decreases in body weight at the end of the study are not noticable in Fig. 3 representing the time-course of body weight development.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The high-dose males showed a minimal, but significant decrease in food consumption from week 30 to the end. However, this was not more than about 5 % of the control.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematologic examinations revealed no clear changes which could be attributed to exposure to methanol.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum biochemical examination showed a small tendency to decrease for alkaline phosphatase, the enzymatic activities of GOT, GPT, LDH and gamma-GTP did not show any differences. Free fatty acid showed lower values in all exposure groups without dose-response relationship. Cholesterol and triglyceride remained unchanged. Changes of other clinical-chemical parameters showed no correlation with the methanol exposure.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis showed no changes suggesting effects from exposure to methanol.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Data of the organ/body weight ratio revealed a dose-related upward tendency in the liver and spleen for females which remained within a 5-% range.

Gross pathological findings:

not examined

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examinations showed a variety of non-tumoral changes in various organs, many of them were infrequent findings which were possibly accidental. Except for swelling of chromophobic cells of the pituitary, there was no findings which could be related to exposure level.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

For tumoral changes, similarly, almost all the findings observed were considered spontaneous ones due to aging.

Key result

Dose descriptor:

NOEC

Effect level:

0.13 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: body weight and food consumption; organ/body weight ratio; swelling of the chromophobic cells of the pituitary

Key result

Dose descriptor:

LOAEC

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: body weight and food consumption; organ/body weight ratio; swelling of the chromophobic cells of the pituitary

Key result

Critical effects observed:

no

Conclusions:

According to the authors, only in the 1.3 mg/L dose-group changes could be considered treatment-related. But based on the minor degree and severity, these changes have no pathological meaning and may be considered as toxicologically irrelevant. Levels of 0.13 mg/L or less (at 20 h/d) did not produce any effect (= NOEC). 1.3 mg/L is adopted as LOAEC, although it may be considered as a NOAEC.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose 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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Comprehensive study programme on three species including metabolic, pharmacokinetic, short-term, long-term, reproductive and carcinogenicity studies.

GLP compliance:

not specified

Species:

monkey

Strain:

Macaca fascicularis

Sex:

not specified

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable, vapor

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

3000 ppm: 20 d
5000 ppm: 5 d and 14 d, respectively
7000, 10000 ppm: 6 d

Frequency of treatment:

21 h/d

Dose / conc.:

13.26 mg/L air (nominal)

Remarks:

corresponding to 10000 ppm

Dose / conc.:

9.31 mg/L air (nominal)

Remarks:

corresponding to 7000 ppm

Dose / conc.:

6.65 mg/L air (nominal)

Remarks:

corresponding to 5000 ppm

Dose / conc.:

3.99 mg/L air (nominal)

Remarks:

corresponding to 3000 ppm

No. of animals per sex per dose:

3000 ppm: 2
5000 ppm: 3
7000 ppm: 1
10000 ppm: 1

Control animals:

yes, concurrent no treatment

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 5000 ppm or more, the animals exhibited reduced movement, weakness, involuntary movement of both hands, vomiting and dyspnea. Food consumption was reduced at concentrations of 5000 ppm or more. The animals exposed to 3000 ppm became used to the methanol atmosphere approximately after 4 days of treatment and recovered activity, movement and appetite, no abnormality was observed after 5 days of treatment. The period of survival at high levels of methanol was considered to be 15 days.

Mortality:

mortality observed, treatment-related

Description (incidence):

The animal at the top dose showed lethargy after 3 treatments, fell into coma and died. The animal exposed to 7000 ppm became critically ill so that it had to be sacrificed. Two animals of the 5000-ppm group had to be killed because of suffering on day 15, one of them died during exposure.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight was normal except for the 10000 ppm exposure group, water consumption was not affected.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals exposed to 7000 and 10000 ppm exhibited a sharp decrease in body temperature and fell into critical condition. These animals showed increased white blood cell counts, possibly due to central nervous disturbance and myocardial disorder. Decreased pH-values were observed were from animals exposed to 500 ppm and more, especially above 7000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No changes in clinical chemistry parameters were observed, except for increased alkaline phosphatase levels at 7000 ppm. Increased neutral lipids and suggesting accelerated lipid metabolism and fatty degeneration were detected in animals exposed to 5000 and 7000 ppm. Mild disturbance of liver function including signs of bile congestion was noted.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes in urine glucose or ketone body were found.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Immediately after different levels of methanol exposure, animals presented common manifestations: they were restless, moving around in the cage, and had frequent blinking and yawning.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nervous system:
Degeneration of bilateral symmetrical putamen, caudate nucleus and claustrum, associated with severe edema, was observed after exposure to 7000 and 10000 ppm and in one of the animals exposed to 5000 ppm. Histological characteristics of these changes included degeneration and disappearance of nerve cells. At 5000 ppm or more, severe edema was also observed in the neighboring cerebral white substance and white substance around the claustrum, associated with necrosis of the basal ganglions.In animals exposed to 3000 ppm slight changes of the same kind were recognized, but no necrotic changes of basement tissues were detected. No significant changes were observed in any regions of the ophthalmencephalon, such as retina, optic nerve, corpus geniculatum and calcinarius.

Viscera:
Marked changes were found in the liver, consisting of slight but clear fatty degeneration of liver cells at 3000 ppm and acute toxic liver necrosis at 5000 ppm. Exposure to 7000 and 10000 ppm caused severe hepatic cell injury in all cases. Vacuolar degeneration was found in the kidneys of animals exposed to 5000 ppm, after exposure to 7000 and 10000 ppm increased mesangium cells were observed in the glomerulus. Methanol exposure did not cause any direct injury to myocardic cells, however, secondary effects of other abnormalities due to methanol inhalation cannot be neglected. No direct effects on lung, thyroid, trachea, stomach, small intestine, pancreas, spleen, aorta, ovary, uterus, or urinary bladder was observed.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

LOAEC

Effect level:

3.99 mg/L air (nominal)

Sex:

not specified

Basis for effect level:

other: clinical signs; histopathology (liver, CNS)

Key result

Critical effects observed:

no

Blood levels of methanol and formate after exposure to methanol:

	3000 ppm	5000 ppm
Methanol	80 mg/L	5250 mg/L
Formate	30 mg/L	1210 mg/L

Cross-reference 4

Reason / purpose for cross-reference:

reference to same study

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

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 LOAEC with a NOAEC 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 NOAEC 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.

Conclusions:

This study served as basis for risk assessment by Vyskocil and Viau (2000): A 1-h reference concentration (RfC) was derived to be at about 100 mg/m³, taking into account sensitive people. A very conservative chronic RfC was obtained and proposed at 0.38 mg/m³, based on the assumption that 0.013 mg/L has to be used as NOAEL and 0.13 mg/L as the LOAEL for "neurotoxic effects". This appears to be not in compliance with the authors´ observations and is not suggested in the report.

Cross-reference 5

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

Special subacute to chronic inhalation test

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The study was designed to investigate the effect of repeated methanol inhalation for various time periods (including recovery phases) in monkeys.

GLP compliance:

not specified

Limit test:

no

Species:

monkey

Strain:

Macaca fascicularis

Sex:

not specified

Route of administration:

inhalation: vapour

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

Duration of treatment / exposure:

see "any other information on materials and methods"

Frequency of treatment:

21 hours/day

Dose / conc.:

1.3 mg/L air (nominal)

Remarks:

corresponding to 1000 ppm

Dose / conc.:

2.7 mg/L air (nominal)

Remarks:

corresponding to 2000 ppm

Dose / conc.:

4 mg/L air (nominal)

Remarks:

corresponding to 3000 ppm

Dose / conc.:

5.3 mg/L air (nominal)

Remarks:

corresponding to 4000 ppm

Dose / conc.:

6.7 mg/L air (nominal)

Remarks:

corresponding to 5000 ppm

No. of animals per sex per dose:

2 to 4

Control animals:

yes, concurrent no treatment

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

not examined

Food efficiency:

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Degenerative changes in the visual system were evident after exposure to 4.0 mg/L and higher: atrophy of the optic nerve and reduction in myelinated fibers. Recovery was unequivocal at exposures below 1.3 and 4.0 mg/L, but at 4.0 mg/L, there appeared to be a trend of these lesions to progress, although -due to the low number of animals- this could also reflect individual variability according to the authors

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lesions of the liver occurred in all dose groups with round-cell infiltration and fibrosis noted in dose-related manner. They were still present histologically after the recovery phase. Changes of the kidneys were observed in all groups: hyalinisation of glomeruli, cell infiltrations into the renal tube stroma. These effects were no longer noted after recovery.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the concentration of 5.3 and 6.7 mg/L, irreversible lesions/degenerations appeared to be likely in the basal ganglions of the cerebrum, because they did not subside within the recovery period, while after 4.0 mg/L (20 d exposure) the slight necrotic degenerations were not progressive as evidenced after recovery. The increase of responsive astroglia seen in the cerebral white substance was still present after recovery from 4.0 mg/L exposure.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/L air (nominal)

Sex:

not specified

Basis for effect level:

other: observed effects were not progressive as evidenced after recovery

Key result

Dose descriptor:

LOAEC

Effect level:

4 mg/L air (nominal)

Sex:

not specified

Basis for effect level:

other: increase of responsive astroglia seen in the cerebral white substance; degenerative changes in the visual system

Key result

Critical effects observed:

no

Conclusions:

At 1.3 mg/L (7 months exposure) some irreversibility of degenerative changes (mild fibrosis presumably following fatty degeneration) unrelated to the recovery period was noted in the liver (p.54) [see also p. 27: chronic study]. These effects were histologically small and, therefore, of little pathological relevance. All other effects -if there were any at all- were not persistent and not considered pathologically relevant at that level, but were significantly more pronounced at 4.0 mg/L and higher after shorter exposure (20 d), associated with eventual lack of recovery. Between 2.7 and 4.0 mg/L appears to be a threshold concentration where methanol-induced neurotoxic effects may become biological relevant (p. 35).

Cross-reference 6

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: inhalation

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Not all parameters mentioned in the guideline were investigated.

Principles of method if other than guideline:

According to OECD Guideline for Chronic Toxicity, Oct. 1980. Comprehensive study programme on three species (rat, mouse, monkey) including metabolic, pharmacokinetic, short-, long-term, reproduction and carcinogenicity studies.

GLP compliance:

not specified

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Housing: individually in wire-mash cages attached to the inhalation chamber
TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 6 wks
- Weight at study initiation: approx. means: males 23 g, females 19 g (taken from graph)
- Fasting period before study: no
- Housing: individually
- Diet (e.g. ad libitum): solid chow (CRF-1, Charles River Japan Inc.) ad libitum
- Water (e.g. ad libitum): sterile-filtered and UV-irradiated tap water ad libitum
- Acclimation period: 5 days of quarantine + 6 days in inhalation chamber


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body exposure chambers (Hazleton 1000 Inhalation Exposure Chamber, 2.5 m³ inner volume)
- Method of holding animals in test chamber: individual rooms in stainless steel wire mesh inhalation cages (60 rooms) installed in the chamber
- Source and rate of air: external air
- Method of conditioning air: successively passed through a medium performance, a high performance, and an activated carbon filter, temperature and humidity regulated
- System of generating particulates/aerosols: total vaporizer, fed by a microquantification pump regulated by a feed back mechanism coupled to a methanol gas analyzer, which measures the concentration in the chamber
- Temperature, humidity, pressure in air chamber: 24±2°C, 55±15%
- Air flow rate: no data
- Air change rate: 15/h
- Method of particle size determination: not applicable, vapor


TEST ATMOSPHERE
- Brief description of analytical method used: Photometric determination by an infra-red spectrophotometer
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical values were close to nominal ones, no further data.

Duration of treatment / exposure:

18 months

Frequency of treatment:

continuously, approx. 19 h/d (10436 - 10550 h (males), 10573 - 10642 h (females))

Post exposure period:

none

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:

52 males, 53 females

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Exposure levels selected on the basis of a 4-wk preliminary exposure study

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day, 5-6 days/wk
- Cage side observations included: mortality, abnormal appearance and behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once per week

FOOD CONSUMPTION: Yes
Food consumption during a week by 2 animals was measured on 24 animals/sex/group, daily food consumption per animal was then calculated.
- Time schedule: weekly during first 13 wks of exposure, monthly thereafter

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes (grossly visible signs)
- Time schedule for examinations: weekly
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all
- Parameters examined: erythrocyte count, leukocyte count, hemoglobin concentration, platelet count, hematocrit value, differential leukocyte count


CLINICAL CHEMISTRY: Yes, from about 20 animals/sex/group
- Time schedule for collection of blood: at necropsy
- Animals fasted: No
- How many animals: all
- Parameters examined: blood methanol levels, GOT, GPT, LDH, ALP, urea nitrogen, glucose, total cholesterol, calcium, inorganic phosphorus, triglyceride, total protein, A/G ratio


URINALYSIS: Yes, from about 20 animals/sex/group
- Time schedule for collection of urine: on a day shortly before sacrifice, not further specified
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: glucose, pH, protein, ketones, bilirubin, occult blood, urobilinogen, intensity of coloring


NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Organ weight, organ/body weight ratio
-Time schedule: at sacrifice of scheduled animals
- Organs: brain, heart, lung, liver, kidney, spleen, testis

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, macroscopic changes of each organ
HISTOPATHOLOGY: Yes:
Brain, Pituitary, Thyroid, Heart, Lung, Trachea, Liver, Thymus, Kidney, Spleen, Pancreas, Adrenal, Testis, Prostate, Seminal vesicle, Ovary, Uterus, Vagina, Urinary bladder, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Femur and bone-marrow, Mesenteric lymph node, Salivary glands (parotid, submaxillary, sublingual), Sciatic nerve, Eyes, Lacrimal gland, Optic nerve, Muscle, Spinal cord, Mammary gland, Hilar lymph node, Submaxillary lymph node, Paranasal cavity, Pharynx, Larynx, Skin, Any other tissues with lesions.

Statistics:

All data obtained were analyzed by t-test, Fischer's exact test or Armitage's chi²-test.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities of the general state could be observed in treated animals.

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival was high without significant differences as compared to the control, mortality ranging between no deaths and 7.5% deaths by the end of the study: 0% (1000 ppm males, 10 ppm females), 2% (controls), 6% (10 and 100 ppm males) and 7.5% (100 and 1000 pm females).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight was somewhat increased in treated group, significantly in 1000 ppm males, between months 6 through 12 in males and months 9 through 12 in females.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of a treatment-related effect in blood parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of a treatment-related effect in clinical parameters. Biochemical examination of plasma gave abnormal values for animals which were found to have tumoral changes at histopathological examination

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of a treatment-related effect in urinalysis parameters.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

For males, the mean testis weight of the 1000 ppm group was lower than that of controls, significant differences were found for absolute value and ratio to body weight due to severe testis atrophy in one male. For females, the kidney weight of the 1000 ppm group was significantly higher than in controls, but there was no significant difference for its ratio to body weight. There were no significant differences for any other organs in the mid- and high-dose groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Higher incidence was noticed in such changes as the swelling of spleen, node in the lung, node in the liver, swelling of preputial gland, ovarian cyst and swelling of uterus. Among these changes, the node in the liver and ovarian cyst were observed with significantly higher incidence, the swelling of spleen with significantly lower incidence, in females in the high-dose group. These changes, however, did not show clear dose-dependency and were considered to be accidental.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Non-neoplastic organ findings were similar to those commonly found in the control animals of 12 months.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Individual tumor frequencies showed no significant differences between all groups; there was no difference in the severity of tumors between the groups. The tumors exclusively observed in the 1000-ppm group were all within the spontaneous range and included fatty cell tumor at the submaxillary lymph node (1/52 males), chromophobe adenoma of the pituary gland (3/52 females), adrenal pheochromocytoma (1/52 females), dermal fibrosarcoma (1/52 males), and meningeal sarcoma (1/52 females) vs. 0/52 in all other groups. The spontaneous liver-tumors rate naturally high in this species was not increased by methanol treatment.

Other effects:

no effects observed

Description (incidence and severity):

The blood levels of methanol and formate were not measured.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 1.3 mg/L air

Sex:

male/female

Key result

Critical effects observed:

no

Exposure per day was 19 h, which implies that prolonged steady-state blood levels were reached which even may have been higher than in studies using the same exposure concentration, but shorter exposure times.

CONCLUSION

The result gave no evidence of a cancerogenic potential of methanol in mice.

Cross-reference 7

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: inhalation

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Not all parameters mentioned in the guideline were investigated

Principles of method if other than guideline:

According to OECD Guideline for Chronic Toxicity, Oct. 1980. Comprehensive study programme on three species (rat, mouse, monkey) including metabolic, pharmacokinetic and short-, long-term, reproduction and carcinogenicity studies.

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Housing: individually in wire-mash cages attached to the inhalation chamber
TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 6 wks
- Weight at study initiation: approx. means: males 120 g, females 100g (taken from graph)
- Fasting period before study: no
- Housing: individually
- Diet (e.g. ad libitum): solid chow (CRF-1, Charles River Japan Inc.) ad libitum
- Water (e.g. ad libitum): sterile-filtered and UV-irradiated tap water ad libitum
- Acclimation period: 1 wk of quarantine + 6 days in inhalation chamber


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body exposure chambers (Hazleton 1000 Inhalation Exposure Chamber, 2.5 m³ inner volume)
- Method of holding animals in test chamber: individual rooms in stainless steel wire mesh inhalation cages (24 rooms) installed in the chamber
- Source and rate of air: external air
- Method of conditioning air: successively passed through a medium performance, a high performance, and an activated carbon filter, temperature and humidity regulated
- System of generating particulates/aerosols: total vaporizer, fed by a microquantification pump regulated by a feed back mechanism coupled to a methanol gas analyzer, which measures the concentration in the chamber
- Temperature, humidity, pressure in air chamber: 24±2°C, 55±15%
- Air flow rate: no data
- Air change rate: 15/h
- Method of particle size determination: not applicable, vapor


TEST ATMOSPHERE
- Brief description of analytical method used: Photometric determination by an infra-red spectrophotometer
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical values were close to nominal ones, no further data.

Duration of treatment / exposure:

24 months

Frequency of treatment:

continuously, 20 h/d (14255 - 14323 h (males); 14407 - 14468 h (females))

Post exposure period:

none

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:

52

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Exposure levels selected on the basis of a 4-wk preliminary exposure study

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day, 5-6 days/wk
- Cage side observations included: mortality, abnormal appearance and behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once per week

FOOD CONSUMPTION: Yes
Food consumption during a week by 2 animals was measured on 24 animals/sex/group, daily food consumption per animal was then calculated.
- Time schedule: weekly during first 13 wks of exposure, monthly thereafter

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes (grossly visible signs)
- Time schedule for examinations: weekly
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all
- Parameters examined: erythrocyte count, leukocyte count, hemoglobin concentration, platelet count, hematocrit value, differential leukocyte count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: No
- How many animals: all
- Parameters examined: blood methanol levels, GOT, GPT, LDH, γ-GTP, ALP, blood urea nitrogen, glucose, total cholesterol, calcium, inorganic phosphorus, triglyceride, total protein, A/G ratio, sodium, potassium, chloride


URINALYSIS: Yes
- Time schedule for collection of urine: on a day shortly before sacrifice, not further specified
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: glucose, pH, protein, ketones, bilirubin, occult blood, urobilinogen, intensity of coloring


NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Organ weight, organ/body weight ratio
-Time schedule: at sacrifice of scheduled animals
- Organs: brain, pituitary, thyroid, thymus, heart, lung, liver, kidney, spleen, adrenal, testis (ovary)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, macroscopic changes of each organ
HISTOPATHOLOGY: Yes:
Brain, Pituitary, Thyroid, Heart, Lung, Trachea, Liver, Thymus, Kidney, Spleen, Pancreas, Adrenal, Testis, Prostate, Seminal vesicle, Ovary, Uterus, Vagina, Urinary bladder, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Femur and bone-marrow, Mesenteric lymph node, Salivary glands (parotid, submaxillary, sublingual), Sciatic nerve, Eyes, Lacrimal gland, Optic nerve, Muscle, Spinal cord, Mammary gland, Hilar lymph node, Submaxillary lymph node, Paranasal cavity, Pharynx, Larynx, Skin, Any other tissues with lesions.

Statistics:

All data obtained were analyzed by t-test, Fischer's exact test or Armitage's chi²-test.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities of the general state could be observed in treated animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

19.2 to 34.6 % of males and 32.7 to 40.4 % of females died or had to be sacrificed in extremis. There were no significant differences from the control groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight was somewhat but not significantly retarded between weeks 51 through 72 in the high-dose females (1000 ppm).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A significant inhibition was noted for high-dose males from week 30 to 52.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not reported.

Haematological findings:

no effects observed

Description (incidence and severity):

No changes were evident in haematological parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No changes were evident in biochemical parameters.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinanalysis revealed a significant increase in glucose in 1000ppm-males, and in females a significant decrease in pH (1000 ppm) and increase in bilirubin (100 and 1000 ppm), but individual values were within normal range.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

None of the organs examined showed significant differences in absolute weight or organ/body weight ratios.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Changes showing significant differences included an increase of nodes in the lung in high-dose males, coinciding with papillary adenoma or adematoid. Further changes concerned differences in depression of brain for mid-dose males, nodes in the liver and swelling of the pituitary and thyroid for females, they were considered to be accidental.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Non-neoplastic findings were similar to those commonly found in the control animals of 24 months. Significant diferences were observed for the kidney of both sexes, the lung of males, and all organs except adrenal of females in the low- and mid-dose groups. They were all considered not treatment-related.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no dose-dependence of tumour development for papillary adenomas, adematoids and the sum of both in males. The spontaneous tumor rate of the Fischer strain in this laboratory and in the published literature was in the range of 14 %. Therefore, the tumours are unlikely to be treatment-related.

No clear dose-dependence was noted for adrenal chromaffinomas in females, but at the highest dose the number of animals with this kind of tumors was significantly increased and was higher than the spontaneous rate published for the Fischer strain (3.2 - 3.5 %) [Note: not clear whether the given data relate only to females or to both sexes. It should be noted that the spontaneous rate of male rats was high in this test compared with that of females.]

A higher incidence of hyperplastic changes in the testes was observed in high-dose males (19.2 %), but no conclusions on dose-dependence could be made as only control and high-dose males were examined.

Overall, tumor frequencies showed no significant differences between all groups. There was no evidence of an increase in liver tumors. Specific tumours appeared at a somewhat higher incidence in high-dose groups of both sexes:
- papillary lung adenomas in males (without dose relationship and not statistically significant: 6/52 vs. 1/52 in the control ), but not in females
- adrenal phaeochromocytomas in females (based on Fisher´s Exact Test: not statistically significant: 7/51 vs. 2/50), but not in males
- 3/52 and 5/52 metastatic (transition) tumors of common origin in males and females, respectively (originated from tumours prone to metatasis). These events having emerged after week 79 and 104 were considered incidental and not treatment-related.

Other effects:

no effects observed

Description (incidence and severity):

The blood levels of methanol were measured but not documented in detail (reported to be similar to those found in the two-generation study). Blood dose levels in the low-dose (10 ppm) and mid-dose (100 ppm) group were almost comparable to those in the control group. In the high-dose group (1000 ppm), however, they were 54 ppm for males and 88 ppm for females. Formate was not measured. Note: Exposure per day was 20 h, which implies that prolonged steady-state blood levels were reached which even may have been higher than in studies using the same exposure concentration, but shorter exposure times.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 1.3 mg/L air

Sex:

male/female

Key result

Critical effects observed:

no

NEOPLASTIC CHANGES:

Incidences (number of tumor-bearing animals/total animals):

Males	0	0.013	0.13	1.3 mg/L
A	1/52	5/50	2/52	6/52
B	4/52	1/50	5/52	4/52
A + B	5/52	6/50	7/52	10/52
C	7/52	--	--	4/41
D	4/52			10/52

-- = not examined

A) Papillary adenomas (males)

B) Adematoids = hyperplastic changes (pre-neoplastic, lung) (males)

C) Adrenal chromaffinoma (males)

D) Testis hyperplastic changes (males)

Females	0	0.013	0.13	1.3 mg/L
A	2/52	--	--	2/52
B	3/52	--	--	1/52
A + B	5/52	--	--	3/52
C	2/50	3/51	2/49	7/51

-- = not examined

A) Papillary adenomas (females)

B) Adematoids = hyperplastic changes (pre-neoplastic) (females)

C) Adrenal chromaffinoma (females)

CONCLUSION

The results gave no evidence of a clearly cancerogenic potential of methanol in rats. The biological significance of the increase in the incidence of the phaeochromocytomas in the aged female high-dose rats is considered to be low.

Cross-reference 8

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, also largely meeting current standards, sufficient documentation, acceptable for assessment.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Exposure and expression period combined

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

no information given

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

15.8, 31.7, 47.4 and 63.3 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (Eagle's MEM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: MNNG, without S9, 0.29 and 0.59 µg/mL

Details on test system and experimental conditions:

DURATION
- Exposure duration: 6 days
- Expression time (cells in growth medium): combined with 6 days exposure
- Selection time (if incubation with a selection agent): after 6 days

SELECTION AGENT (mutation assays): 8-Azaguanin, 6-Thioguanin, Ouabain

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (colony formation)

Evaluation criteria:

Significant increase in V79 cells resistant to 8-Azaguanine, 6-Thioguanine or Ouabain.

Statistics:

Calculation of mean mutation frequency ± S.D. per 10exp+6 surviving cells.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

63.3 mg/mL (approx. 70% inhibition of colony formation)

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test substance induced no increases in mutant frequency in gene mutation to drug resistance vs. negative control, whereas the positive control DMN produced increases in dose-related manner in the presence of metabolic activation (S9 mix), and MNNG in the absence of metabolic activation.

Maximum mutation frequency of V79 cells (per 10exp+6 survival cells, mean ± SD) for resistance towards 6-TG, 8-AG and Ouabain after methanol treatment:

	Control		Test substance (mg/mL)
Selectant	-S9	+S9	-S9	+S9
6-Thioguanine	0.70±1.79	0.94±2.18	1.55±3.08 (47.4 mg/mL)	0.84±2.21 (31.7 mg/mL)
8-Azaguanine	23.42±8.70	24.29±7.30	22.34±7.39 (31.7 mg/mL)	20.05±13.01 (31.7 mg/mL)
Ouabain	1.23±2.23	0	2.64±2.79 (47.7 mg/mL)	0.11±0.36 (47.7 mg/mL)

Conclusions:

negative

Executive summary:

The test substance induced no increases in mutant frequency in gene mutation to drug resistance vs. negative control, both in the presence and absence of metabolic activation, whereas the positive control DMN produced increases in dose-related manner in the presence of metabolic activation (S9 mix), and MNNG in the absence of metabolic activation.

Data source

Reference

Reference 1

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 2

Reference Type:

publication

Title:

Long-term effects of methanol vapor at low concentration.

Author:

Takeda, K. and Katoh, N.

Year:

1988

Bibliographic source:

Proc. of the 8th Int. Symp. Alcohol Fuels, 1051-1056, Tokyo, Japan

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

- limited documentation; copulation time was too long (21 days); not all parameters mentioned in the guideline were investigated

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

Methyl alcohol

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 8 weeks
- Diet: Solid Chow for rat (CRF-1, Charles River Japn Inc.)
- Water: sterilised and filtrated water (ad libitum)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2 °C
- Humidity (%): 55 ± 15 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: multi-stage inhalation chamber (Hazleton 1000 exposure chamber)
- Temperature, humidity in air chamber: 24 ± 2 °C; 55 ± 5 %


TEST ATMOSPHERE
- Nominal exposure levels were prepared by generating methanol gas and then mixing it with fresh air.
- A methanol gas analyser measured the concentration in the chamber.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 21 d
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Other: The pairs without evidence of insemination within 21 d were again cohabited with untreated animals (2nd mating) to determine the fertility of each animal, in this case without exposure (p.186).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical concentration values of methanol were close to nominal ones (the monthly variation remained less than 5 %).

Duration of treatment / exposure:

F0: 103 -108 d
F1: 61 -62 d and 145 -153 d
F2: 54 -56 d
further informations see "any other information on materials and methods"

Frequency of treatment:

continuously

Details on study schedule:

- F1 parental animals not mated until 12 weeks after selected from the F1 litters.
- Female F1 animals were examined for sexual cycle at 12-weeks or thereafter and mated with males of the same group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.013 mg/L air

Remarks:

corresponding to 10 ppm

Doses / concentrations 2

Dose / conc.:

0.13 mg/L air

Remarks:

corresponding to 100 ppm

Doses / concentrations 3

Dose / conc.:

1.3 mg/L air

Remarks:

corresponding to 1000 ppm

No. of animals per sex per dose:

30 (F0 generation)
Additionally, 15 animals were reared for a second mating.

Control animals:

yes, sham-exposed

Examinations

Parental animals: Observations and examinations:

Observations of F0 and F1 parental animals.
CAGE SIDE OBSERVATIONS:
- clinical signs, mortality, any sign of abortion and premature delivery
- Time schedule: at least once a day, 5 days a week
BODY WEIGHT:
- Time schedule for examinations: animals were weighed weekly: day 0, 7, 14 and 20 of gestation and day 0, 4, 7, 14 and 21 of delivery
FOOD AND WATER CONSUMPTION::
- consumption measured by cage (on the same days as body weight measurements)
OTHER:
- Generally sexual cycle, mating time, fertility, pregnancy rate were documented. During the lactation period, maternal animals were observed for nursing behavior including lactation, nest building and presence/absence of pup-eating.

Sperm parameters (parental animals):

Histological examination of morphology of sperms was not included.

Litter observations:

Observations of F1 and F2 litters.
Litters were examined on the day of birth for live pups, dead pups, sex and any external abnormalities. The observations were done daily until weaning and thereafter 5 days a week.
Each litter was weighed on day 0 and 4 (before reduction) of birth by sex and respective mean value calculated. After adjustment of litter size, pups were weighed individually on day 4, 7, 14 and 21. From weaning to week 14 of birth, the measurements were done weekly.

All surviving pups were observed for post-natal morphological differentiation indices: pinna unfolding, eruption of incisors, open eyes, descensus testis (males), vagina opening (females).
As for movement function test, all surviving pups after adjustment of litter size were tested for righting on a surface, ipsilateral flexor reflex, pinna reflex, auricular startle response, visual recognition response, pain response, corneal reflex and suspension abililty on a particular day before weaning. Also emotional tests, learning ability tests, and movement coordination tests were included.

In 9-week old F1 pups, blood methanol was measured, but not formate (p. 191).

Postmortem examinations (parental animals):

Examinations of F0 and F1 parental animals.

After mating all males were necropsied, and testes, epididymis, seminal vesicle and postate gland were removed and preserved.

After 2 weeks of rearing, the females at the 2nd mating were necropsied and examined for pregnancy status. After termination of mating, the not inseminated females were necropsied and the ovary, uterus and bagina were preserved.
21 days after delivery, all dams were necropsied and examined for implantation. The vagina, uterus and ovary were preserved.

Any organ with any abnormality was subjected to a histopathological examination, if necessary.

26 days after evidence of insemination, females which had not yet delivered were necropsied and subjected to the same examinations as the above.

Postmortem examinations (offspring):

Examinations of F0 and F1 litters.

After termination of movement function tests, pups were sacrificed and necropsied.
The pups which were selected for examination and not used for the movement function test were necropsied on a day of the same age at 8 weeks old or thereafter and principal organs were weighed.

Statistics:

All data obtained were analysed by t-test, Fischer´s exact test, U-test of Mann-Whitney or Armitage´s chi²-test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related alterations in general observations.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences for body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences for food consumption.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There were no differences for water consumption.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormalities were observed in findings on nursing behavior.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

None of the fertility indices including sexual cycle, days needed for insemination, insemination rate and pregnancy rate showed statistically significant differences.
No abnormalities were observed in findings on delivery and nursing behavior and necropsy data of F0 animals.

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P1)

Key result

Dose descriptor:

NOAEC

Remarks on result:

not measured/tested

Target system / organ toxicity (P1)

Key result

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In male pups of the 1.3 mg/L group, post-natal morphological differentiation appeared to be influenced with respect to the descensus testis occurring 0.5 to 1 d earlier (see same effect in F2 generation)[not mentioned by Takeda and Katoh, 1988]: This time-dependent parameter was evaluated by relating the completion of downward migration of the testes (final length of the gubernaculum reached) to the post-natal body-weight gain (The more reliable body length was not available):
In the F1 pups derived from the 1.3 mg/L group (108 males), this process was completed within 16 through 20 post-natal days with the climax at day 17 and 18 (32 and 39 %, respectively), while in the respective control (113 males), descent was complete from 16 through 21 days with the maximum at day 19 (32 %), but also relatively high percentages on the days before and after: day 18 (22%), day 17 (19%), day 20 (18%).

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

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 examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

None of the fertility indices including sexual cycle, mating time, fertility and pregnancy rate showed a significant difference from untreated F1 controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative brain weights were significantly lowered in the high-dose groups of either sex at an age of 8 and 16 weeks. This was still found in females necropsied after 24 weeks. Also other organs showed slight shifts in weights: thymus, pituitary (lower), heart, lung, liver (higher).

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

no histopathological manifestations; no effects on testes or ovaries reported

Other effects:

no effects observed

Description (incidence and severity):

There were no significant differences in functional tests (movement, emotion, learning) as compared with the control or the other groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

0.13 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

2

Key result

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

As in F1 males, an apparently dose-related earlier descensus testis was noted after 1.3 mg/L exposure: F2 (94 males) on day 16 (42%), day 17 (40%), day 18 (15%) vs. control (91 males) on day 16 (10%), day 17 (39%), day 18 (31%), day 19 (14%) (p. 200).After 0.13 mg/L, "descensus testis" in male F2-progeny was about 0.5 d earlier than in male control F2 pups (p. 195). Detailed data not specified and not addressed under "Discussion" in the study report.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

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 examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights showed similar tendencies as found in the F1-generation.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

no histological changes; no effects on testes or ovaries reported.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

0.13 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

2

Key result

Dose descriptor:

LOAEC

Generation:

F2

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Blood levels of methanol measured in the F1-offsprings (age 9 weeks) (NEDO, 1987, p. 191):

controls (baseline): approx. 2 - 3 mg/L

0.013 mg/L methanol: approx. 3 - 3.5 mg/L

0.13 mg/L: approx. 1 - 4.2 mg/L

1.3 mg/L: approx. 53 (males)-100 (females) mg/L

There are no data on formate.

Note: Exposure per day was 20 h, which implies that prolonged steady-state blood levels were reached which even may have been higher than in studies using the same exposure concentration, but shorter exposure times.