Reaction mass of methyl acetate and methanol

Endpoint summary

Description of key information

The reaction mass of methyl acetate and methanol is assessed on the basis of the individual constituents methyl acetate and methanol using a read-across approach from the supporting substances (structural analogue or surrogate).

 

Repeated dose toxicity: inhalation

Methyl acetate

In a subacute repeated dose inhalation study according to OECD guideline 412, the NOAEC of methyl acetate was found to be 1057 mg/m³ in rats.

Methanol

In four different repeated dose inhalation studies (Klimisch score 2) with methanol, the NOAECs were 6660 mg/m³ in rats after subacute exposure, 1060 mg/m³ in rats in after subchronic exposure, 1300 mg/m³ in monkeys after 7 months exposure and 13 mg/m³ in monkeys after chronic exposure. Additionally, the LOAEC in a subacute inhalation study (Klimisch score 2) was 3990 mg/m³ in monkeys.

 

According to (EC) No 1907/2006, Annex VIII, 8.6.1, column 1, a test for short-term toxicity by repeated application to male and female animals of a species must be conducted, taking into account the route of exposure expected for humans. Subchronic toxicity testing according to Annex IX, 8.6.2, column 2 may be omitted if the substance rapidly cleaves and the cleavage products are well examined.

Therefore, as the most likely route of human exposure to methyl acetate and methanol is inhalation, repeated oral and dermal toxicity studies are waived. As methyl acetate hydrolyses into methanol and acetic acid and the chronic hazard profile is well known for the hydrolysis products, a subchronic toxicity study with methyl acetate does no need to be conducted for any route. A long-term repeated toxicity study (>/= 12 months) is not proposed for methyl acetate as none of the conditions of Annex X, 8.6.3., column 2 are met.

 

Conclusion

Based on GHS criteria and based on the more critical data obtained for methanol, the reaction mass of methyl acetate and methanol is not classified for specific target organ toxicity (repeated dose).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

other: Read-across, see cross-reference

Justification for type of information:

Please refer to IUCLID section 13 for Read Across Justification.

Reason / purpose for cross-reference:

read-across source

Histopathological findings: non-neoplastic:

effects observed, treatment-related

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

13 mg/m³

Study duration:

chronic

Experimental exposure time per week (hours/week):

147

Species:

monkey

Quality of whole database:

Studies well documented, meets generally accepted scientific principles, acceptable for assessment

System:

cardiovascular

Organ:

heart

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity, methyl acetate

Subacute inhalation, rat, RL1

The likely route of human exposure is inhalation. Other routes are not examined.

 

Methyl acetate was tested in a 28-day inhalation study which was performed according to the B.8 method (OECD 412) (HMR; 1999a). Groups of 10 male and 10 female Sprague- Dawley rats received methyl acetate (test substance purity >99.5%) by nose-only inhalation exposure on concentrations of 0, 75, 350 or 2,000 ppm (mean analytical concentrations 79, 335 and 2,018 ppm, equivalent to 0.227, 1.057, 6.040 mg/L) on 6 hours daily, 5 days per weeks.

 

At the high concentration groups, the body weights and food consumptions were decreased in both sexes (males > females) without significant differences in the mean relative food consumption/kg bw/d. The mean values of erythrocyte counts, hemoglobin and hematocrit were increased, while the total counts of leukocytes and lymphocytes were decreased for these groups. Clinical chemistry examinations revealed a dose-related decrease of cholesterol levels gaining significance in both sexes at the high concentrations and in females at all dose groups. Serum calcium concentrations were increased in both sexes at the high concentration. The ALAT activities were slightly, but significantly increased in high concentration females (41 U/L vs. 34 U/L in controls). Females from the high concentration group showed significantly increases in urine volume and decreases in specific weight. Due to the effect on body weights several absolute organ weights were decreased and relative organ weights were increased in high concentration males. Animals from the high concentrations groups had increased adrenal weights in both sexes and decreased thymus weights in females. Slight significant changes of adrenal and thymus weights were also observed in females of the intermediate concentration group. At necropsy, no compound-related macroscopic findings were observed. Histopathologic examinations revealed slight to moderate degeneration and necrosis of the olfactory epithelium (at level 3 out of 4 sections) of mainly all males and females exposed to the high concentration of methyl acetate. Any other treatment-related abnormality was observed in any other organs and in any other dose group. As degeneration of the olfactory mucosa occurred at 2000 ppm, the NOAEL for local effect was estimated to be 350 ppm (1.057 mg/L). The food efficiency was similar in animals from all groups, therefore the reduction of body weights were considered to be related to the reduced food consumption. The toxicological significance of altered adrenal weight and reduced cholesterol levels was considered to be equivocal as no morphological abnormality of the adrenal was observed. It can be interpreted to indicate nonspecific toxic response e.g. due to stress. However a specific response of the adrenal cortex cannot be excluded; data on the serum levels of steroid hormone concentrations were not generated. The red blood changes may indicate hemoconcentration due to treatment-related increased diuresis observed in high concentration females and/or reduced water consumption. This remains uncertain, because of missing data on the water consumption. The increase of ALAT activity in rats is indicative for hepatocellular damage. In absence of any morphological lesion or any other corresponding change this was considered indicative for a minimal dysfunction of liver cells. The weight reduction of thymus and leucocytopenia/lymphopenia is not regarded indicative for an immunosuppressive effect as morphological changes in the thymus or any other immune organ were absent. Overall, there is a concern that diureses, minimal liver cell dysfunction, adrenal weight increase, and reduced serum cholesterol concentrations represented minimal adverse effects due to the methyl acetate treatment.

 

Therefore the NOAEL for systemic effects was also derived to be 350 ppm (1057 mg/m³).

 

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

At the concentration of 350 ppm, slight increases in adrenal weights and slight decreases in thymus weights were observed in females. As there were no histopathological changes in these organs even at the much higher concentration of 2000 ppm, these findings were considered not to be of toxicological relevance).

 

 

Repeated dose toxicity, methanol

Subacute inhalation, rat, RL2

Potential toxic effects of methanol vapours (6 hours exposure/day, 5 days/week). were investigated in a subacute inhalation study similar to OECD guideline 412 in male and female rats. Dose concentrations were 520, 1980 and 5010 ppm. 5 animals per dose were used. Increased frequencies of nasal and eye discharge (lacrimation, mucoid nasal discharge, red nasal discharge, and dried red nasal discharge) were noted in the treated groups. Only mucoid nasal discharge appeared to be dose-related. No mortality was observed, there were no treatment-related effects on body weight and no ocular abnormalities were noted at the terminal examination of the high-dose group. No effects were observed in clinical biochemistry. Relative spleen weights were significantly (p ≤ 0.05) increased in female rats exposed at 1980 ppm (not at 5010 ppm). No other organ weight effects were noted. No (neuro-)pathological findings were observed and no treatment-related histopathological effects were noted. The NOAEC was thus found to be 6660 mg/m³.

 

 

Subchronic inhalation, rat, RL2

Potential toxic effects of methanol vapours on the morphology of the testes in normal or folate-reduced rats were investigated in a subchronic inhalation toxicity study (20 hours exposure/day, 7 days/week) in male rats. Dose concentrations were 50, 200 and 800 ppm. 11 to 12 animals (10 months old) for three exposure levels, and 8 animals (18 months old) for a 800-ppm group were used. The testes-to-body weight ratio of rats exposed up to 800 ppm for up to 13 weeks (20 h/d, 7 d/wk) was not different from control animals, irrespective of  the nutritional state, normally fed or folate-deficient. The testicular morphology of rats exposed up to 800 ppm methanol for up to 13 weeks (20 h/d, 7 dw) was not different from control animals. A greater incidence of testicular degeneration was seen only in 18 month old, folate-deficient rats exposed to 800 ppm methanol for 13 weeks (20 h/d, 7 d/wk). The results are equivocal. 800 ppm (1.06 mg/L/1060 mg/m³), the top level, can be established as subchronic NOAEC based on testicular histopathology.

 

 

Subacute to chronic inhalation, monkey, RL2

Potential toxic effects of methanol vapours (21 hours exposure/day). were investigated in a special subacute to chronic inhalation test programme including recovery phases in monkeys. The 1000 ppm group (2 animals) was exposed for 7 months followed by recovery periods of 30 days and 6 months. The 2000 and 3000 groups were exposed for 20 days followed by recovery periods of 30 days, 6 months and 10 months each. The 5000 ppm group (2 animals) was exposed for 6 days at 5000 ppm followed by another 6 days at 4000 ppm after which one moneky recovered for 30 days and the other for 4 months. Degenerative changes in the visual system were evident after exposure to 3000 ppm and higher: atrophy of the optic nerve and reduction in myelinated fibers. Recovery was unequivocal at exposures below 1000 and 3000 ppm but at 3000 ppm, 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. 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. At the concentration of 4000 and 5000 ppm, 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 3000 ppm (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 3000 ppm exposure. At 1000 ppm (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 3000 ppm and higher after shorter exposure (20 d), associated with eventual lack of recovery. Between 2000 and 3000 ppm appears to be a threshold concentration where methanol-induced neurotoxic effects may become biological relevant (p. 35). Therefore, the NOAEC was found to be 1300 mg/m³.

 

 

Chronic inhalation, monkey, RL2

Potential toxic effects of methanol vapours (21 hours exposure/day). after chronic exposure of monkeys were investigated. Dose concentrations were 10, 100 and 1000 ppm. 2 monkeys (7 months exposure), 3 monkeys (1 year + 7 months (19 months) exposure) and 3 monkeys (2 years + 5 months (29 months) exposure) were used. The monkeys of the 1000 ppm group showed abnormal scratching of the skin and clinical symptoms such as frequent yawning and nasal discharge. The animals showed normal body-weight gain, food and and water consumption throughout. Examinations of the eye fundus revealed no changes in any group. 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. No significant differences in serum values were seen. However, one monkey in the 100 ppm group showed differences for most liver parameters (total protein, GOT, GPT, total cholesterol, free cholesterol, thymol and glucose). This was not considered treatment-related.

 

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 100 and 1000 ppm groups, in particular in the 100 ppm 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 10 ppm 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 10 ppm 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 100 and 1000 ppm 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 100 ppm and 2 at 1000 ppm 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 1000 ppm along with signs of fibrosis in the hepatic cord more pronounced than at the lower concentration. Sudan-positive granules were observed at 100 ppm and 1000 ppm in the renal tubular epithelium (NEDO, 1987, p. 26/27).

 

Heart: There was an increase in the Sudan-positive granules in heart tissue at 1000 ppm, but not manifested in any morphological lesion of the heart muscle (e.g. fibrosis).

 

In both upper dose groups, the ECG of one of the 100 ppm exposed monkeys and three of the 1000 ppm exposed monkeys showed abnormalities (negative T- or Q-wave changes and flattening of T-wave) which indicated slight myocardial disorder (p. 21).

 

Taking into account slight myocardial effects and slight hyperplasia of the astroglia in the cerebral white substance already noted at 100 ppm, then already 100 ppm have to be defined as LOAEL with a NOAEL of 10 ppm. No clear adverse effects are reported at 1000 ppm. No necrotic effects occurred in the nervous tissue. 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. 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 1000 ppm, which were associated with signs of fibrosis. This effect was still present after recovery (see other entry). Given the small histological effect, the pathological relevance appears to be low, but indicates that long-term exposure to 1000 ppm methanol is on the borderline to toxicologically relevant, histological manifestations. 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 10 ppm 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 1000 ppm methanol. Therefore, the NOAEL for continuous exposure may be established at 1000 ppm, but the low number of animals and limited description do not allow to draw firm conclusions on the apparent neural effect. 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 10 ppm has to be used as NOAEL and 100 ppm as the LOAEL for "neurotoxic effects". This appears to be not in compliance with the authors observations and is not suggested in the report. Thus, the NOAEC for repeated dose toxicity is considered to be 13 mg/m³.

 

 

Subacute inhalation, monkey, RL2

Potential toxic effects of methanol vapours (21 hours exposure/day). were investigated in a subacute inhalation test programme in monkeys. Dose concentrations were 3000 ppm for 20 days (2 animals), 5000 ppm for 5 and 14 days (3 animals), 7000 ppm for 6 days (1 animal) and 10000 ppm for 6 days (1 animal). 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. 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. The body weight was normal except for the 10000 ppm exposure group, water consumption was not affected. 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. 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. No changes in urine glucose or ketone body were found. 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.

 

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. In summary, a LOAEC of 3990 mg/m³ was determined.

 

Conclusion

Based on GHS criteria and based on the more critical data obtained for methanol, the reaction mass of methyl acetate and methanol is not considered to induce profound effects after repeated dose exposure.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The reaction mass of methyl acetate and methanol is assessed on the basis of the individual constituents methyl acetate and methanol. The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Following a worst scenario, methanol, the more critical of both substances, is used for the classification. The experimental studies with animals do not provide clear evidence for the necessity for classification: In primates a potential of methanol to cause adverse health effects was shown, while in rodents only toxicologically irrelevant effects were shown. As a result, the reaction mass of methyl acetate and methanol is not classified for special target organ toxicity, repeated dose under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/217.