Trimethyl orthoacetate

Endpoint summary

Description of key information

Trimethyl orthoacetate is rapidly hydrolysed to methanol (CAS no. 67 -56 -1) and methyl acetate, which further hydrolyses to acetic acid (CAS no. 64 -19 -7) as final reaction product in the presence of water or moisture at pH 4, 7 and 9 (< 2.4 h 50°C). Under neutral (pH 7) and acidic (pH 4) conditions, the half-life was < 1 h. Accordingly, reliable data of the hydrolysis products methanol and acetic acid are used to address the endpoint, which is entirely appropriate to draw conclusions on the repeated dose toxiciy of Trimethyl orthoacetate to mammals. For each hydrolysis product results on the repeated dose toxicity are presented for the most appropriate route of administration, based on respective physical chemical parameters.

1. Methanol

- Repeated dose toxicity: chronic (12 months) study, inhalation (vapour), rat ( Fischer 344/DuCrj) m/f, 3 concentrations (0.013, 0.13 and 1.3 mg/L air, corresponding to 10, 100 and 1000 ppm) (OECD TG 453): NOAEC = 1.3 mg/L air, NOEC = 0.13 mg/L air (nominal, m/f)

- Repeated dose toxicity: chronic (12 months) study, inhalation (vapour), mouse ( B6C3F1) m/f, 3 concentrations (0.013, 0.13 and 1.3 mg/L air, corresponding to 10, 100 and 1000 ppm)

(OECD TG 453): NOAEC = 1.3 mg/L air, NOEC = 0.13 mg/L air (nominal, m/f)

2. Acetic Acid

- Repeated dose toxicity: subacute (8 weeks) study, oral (feed), rat (spontaneously hypertensive rats) male (no guideline followed, non-GLP): NOAEL = 290 mg/kg bw/day (nominal, male)

- Repeated dose toxicity: subchronic (6 months) study, oral (feed), pig (no guideline followed, non-GLP): NOAEL = 450 mg/kg bw/day (nominal)

- Repeated dose toxicity: subacute (4 weeks) study, oral (feed), rat (Wistar, vitamin B-12 deficient) (no guideline followed, non-GLP): NOAEL =3.58% sodium acetate (1.58% acetate moiety) with or without vitamin B12, corresponding to a NOAEL of 2370 mg/kg bw/day for the acetate moiety (nominal, male)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP, non-guideline, animal experimental study. Minor restrictions in design and/or reporting but otherwise adequate for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Feeding study designed to determine whether vitamin B12 is required for the metabolism of odd-carbon fatty acids higher than propionate and of certain branched-chain fatty acids that lead to propionate.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- From mothers who were transferred from a stock ration to a vitamin B12 deficient ration at parturition and continued on the deficient ration during lactation.
- Age at study initiation: Approximately 28 days
- Housing: Individually in cages with raised screen floors
- Diet: 25% protein, vitamin B12 deficient diet ad libitum, with or without the addition of fatty acids
- Fatty acids, when fed, replaced an equal quantity of dextrin in the diet. 1.58% acetate moiety (fed as sodium acetate, 3.58% of ration)
- Vitamin B12, when given, was included in the ration at a level of 5 µg/10 g diet
- Water: ad libitum
- Acclimation period: At least 3 days

ENVIRONMENTAL CONDITIONS: no data
IN-LIFE DATES: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Continuous

Remarks:

Doses / Concentrations:
3.58% sodium acetate
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
equivalent to 1.58% as the acetate moiety
Basis:
nominal in diet

No. of animals per sex per dose:

6 males (without vitamin B12), 7 males (with vitamin B12)

Control animals:

yes, plain diet

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

1.58 other: % based on the acetate moiety

Sex:

male

Basis for effect level:

other: The effect on growth of rats fed diet containing 3.58% sodium acetate, with or without vitamin B12, was not significantly changed when compared to rats fed basal diet only.

Remarks on result:

other:

Remarks:

see "Overall remarks, attachments"

Critical effects observed:

not specified

Acetate and higher even-carbon fatty acids had no effect on such growth. While the 5-, 7- and 9-carbon straight-chain fatty acids depressed growth on the control ration, as did propionate itself, the extent of depression decreased as the carbon chain lengthened, reflecting perhaps a tendency for the higher acids to be partially metabolized by an alternate pathway. The branched-chain acids (isobutyric, 2-methyl butyric and 4-methyl valeric), depressed growth on the control ration but isovaleric acid did not.

Effect of vitamin B12 on growth of rats fed acetic acid

			Average weight gain g (weeks 2-4)
Amount in diet %	# rats per group	Vitamin B12	Basal diet	Basal diet + fatty acid	% change
1.58	6	No	39	45	+15
1.58	7	Yes	125	124	-1

 

Conclusions:

The effect on growth of rats fed diet containing 3.58% sodium acetate (1.58% acetate moiety) in basal diet, with or without vitamin B12, was not significantly changed when compared to rats fed basal diet only. Accordingly, the NOAEL was determined to be 3.58% sodium acetate or 1.58% based on the acetate moiety. These doses correspond to concentrations of 5370 mg/kg bw/day for sodium acetate and 2370 mg/kg bw/day for the acetate moiety (see "Overall remarks, attachments").

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, non- guideline study, published in peer reviewed literature. No restrictions, fully adequate for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 6 spontaneously hypertensive rats were fed diets containing 6% (w/w) acetic acid , 6% (w/w) rice vinegar or control diet for 8 weeks. Blood pressure, heart rate, body weight, food intake and water consumption were measured at weekly intervals. Urine samples were collected every 2 weeks for measurements of volume, sodium, calcium and catecholamine excretion. After 8 weeks, animals were killed and blood samples collected from the aorta. The heart, aorta, kidneys and lungs were removed and the angiotensin I-converting enzyme (ACE) activity was measured.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: spontaneously hypertensive rats

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoshino Laboratory Animals, Japan
- Age at study initiation: 4 weeks old
- Weight at study initiation: not reported
- Fasting period before study: none
- Housing: Metabolic cages (no further details reported)
- Diet: Labo MR stock diet, Nihon Nosan Kogyo KK, was mixed 6% with deionised water (control), acetic acid or rice vinegar and fed ad libitum
- Water: tap water ad libitum.
- Acclimation period: 6 days (fed on control diet)

ENVIRONMENTAL CONDITIONS
- Temperature: 24 ± 2°C
- Humidity: 50 ± 10%
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light (light from 07:00 to 19:00)

IN-LIFE DATES: not reported

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): not reported
- Mixing appropriate amounts with (Type of food): standard laboratory diet mixed 6% (w/w) with deionised water, acetic acid or rice vinegar
- Storage temperature of food: not reported

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
6% (w/w)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
290 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Vinegar extracts are thought to play a role in blood pressure reduction but the main components of commercial vinegar are acetic acid and saccharides. This study aimed to clarify the possibility of the preventative effects of long-term administration of dietary vinegar and pure acetic acid on hypertension using spontaneously hypertensive rats (SHR), a model for human hypertension.
- Rationale for animal assignment (if not random): After acclimatisation to the control diet, 18 animals were divided into 3 groups of 6 animals, so that each group had the same mean bodyweight and blood pressure.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data
- Time schedule: Not specified

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day/rat: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: every other week
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Urine volume, sodium, calcium and catecholamine excretion were examined

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: At termination, blood was taken from the aorta ventralis, EDTA plasma was prepared and analysed, using radioimmunoassays, to detect renin activity, angiotensin II, aldosterone and PGE2

Sacrifice and pathology:

All rats were killed under pentobarbitone anaesthesia (30 mg/kg). The heart, aorta, kidneys and lungs were removed.

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

Other examinations:

Angiotensin I converting enzyme (ACE) activity was measured from the heart, aorta, kidneys and lungs. Enzyme extracts were prepared by chopping each organ into small pieces and homogenising in 50mm Tris HCl (pH 7.9) containing 0.3M NaCl. The suspension was centrifuged and the resulting supernatent fluid was centrifuged again. The pellet was suspended in a 0.1M sodium borate buffer (pH8.3) containing 0.3M NaCl and the suspension was used as the enzyme extract. The ACE activity was assayed according to Kasahara and Ashihara.

Statistics:

All values were expressed as means ± SD. Student's t-test was used to evaluate the significance of any differences in the ACE activity of each organ. An analysis of variance (ANOVA) was used to evaluate differences between the groups; when ANOVA indicated any significant differences between the means, Fisher's PLSD test was used to find which means were significantly different. P< 0.05 was defined as significant.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The average food consumption of the control group was 18.8 ± 2.4 g/day/rat and those of the acetic acid and rice vinegar groups were 19.3 ± 2.6 and 19.1 ± 2.4 g/day/rat, respectively. These food intakes corresponded to the ingestion of about 0.86 mmol/day/rat of acetic acid.

BLOOD PRESSURE
The blood pressure of rats in both experimental groups tended to be lower than the controls after 8 weeks of age and significant differences were observed after 10 weeks of age in both groups. Blood pressure values for the acetic acid and rice vinegar groups were 164 ± 12.4 and 165.2 ± 18.5 mmHg at 11 weeks of age, respectively, while that for the control group was 186.2 ± 7.8 mmHg at the same age; a 21 mmHg decrease in blood pressure was observed. At 13 weeks of age, the end of the feeding period, the rice vinegar group showed a statistically significant decrease in blood pressure of -30 mmHg compared to the control group. At 13 weeks the blood pressure of the acetic acid group was 13 mmHg lower than that of the control group but this did not attain statistical significance.

ACE ACTIVITY AND PLASMA BIOCHEMISTRY
The angiotensin converting enzyme (ACE) activity of the lungs, heart, aorta and kidneys showed no significant differences from the control group. (Table 1).
Plasma renin activity and aldosterone changed significantly with the long term administration of both experimental diets (Table 2). The plasma renin activity of the acetic acid and rice vinegar groups decreased to 9.9 ± 2.2 and 8.7 ± 3.3 ng/mL/hr, respectively, compared to the control group value of 15.0 ± 2. The plasma aldosterone levels of the acetic acid and rice vinegar groups were 101 and 44.7 pg/mL, respectively, while that of the control group was 131 ± 43.5 pg/mL. However, plasma aldosterone in the rice vinegar group was statistically significantly reduced compared to both the acetic acid and control groups.
Plasma angiotensin II was slightly lower than controls in both experimental groups but this was not statistically significant.

URINE ANALYSIS
There was no significant difference in sodium excretion or catecholamine concentrations in any of the groups throughout the experimental period.

Dose descriptor:

NOAEL

Remarks:

bodyweight, clinical symptoms

Effect level:

290 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: a rise in blood pressure was observed in all groups and plasma renin activity was reduced, however these effects were considered not to be adverse in the context of establishing an NOAEL for this study

Critical effects observed:

not specified

Table 1. Angiotensin I-Converting Enzyme Activity in Organs of Spontaneously Hypertensive Rats at 13 Weeks of Age

	Angiotensin I-Converting Enzyme Activity (mU/mg protein)
Group	Lung	Aorta	Heart	Kidney
Control	119 ± 3.3	3.72 ± 1.3	1.06 ± 0.14	5.19 ± 2.0
Acetic acid solution	122 ± 5.2	3.90 ± 0.76	1.06 ± 0.10	6.65 ± 1.6

Feeding of the diet containing acetic acid or rice vinegar started at 5 weeks of age. Results are expressed as the mean and standard errors. There were no significant differences from the control.

Table 2. Effects of Acetic Acid and Vinegar on Blood Indices

Group	Plasma rennin activity (ng/ml•hr)	Plasma angiotensin II(pg/ml)	Plasma aldosterone(pg/ml)
Control	15 ± 2.0	33.8 ± 11	131 ± 43.5
Acetic acid solution	9.9 ± 2.2*	25.2 ± 8.7	101 ± 46.9 r
Rice vinegar	8.7 ± 3.3**	29.2 ± 12	44.7 ± 32.2* r

Feeding of the diet containing acetic acid or rice vinegar started at 5 weeks of age. Results are expressed as the mean and standard errors. Significant difference from control: *p<0.01, **p<0.001. Symbol r indicates that there were significant differences between the acetic acid and rice vinegar groups (p <0.05).

Conclusions:

No adverse effects were observed in male SHR rats with an NOAEL of 290 mg/kg bw/d

Executive summary:

To clarify the possibility of a preventive effect of dietary vinegar on blood pressure, long-term administration of vinegar or the acetic acid to Spontaneously Hypertensive Rats was examined. As a result, it was observed that acetic acid itself, the main component of vinegar, significantly reduced both blood pressure (p<0.05) and renin activity (p<0.01), compared to controls given no acetic acid or vinegar, as well as vinegar. There were no significant differences in angiotensin I-converting enzyme activity in various organs. As for the mechanism of this function, it was suggested that this reduction in blood pressure may be caused by the significant reduction in renin activity and the subsequent decrease in angiotensin II. From this study, it was also suggested that the antihypertensive effect of vinegar is mainly due to the acetic acid in it.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, non- guideline study, published in peer reviewed literature. Predates implementation of GLP and/or development of guidelines but otherwise acceptable for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline available

Principles of method if other than guideline:

Young pigs (from a single litter) were divided into groups of 2 and fed basal diet (control group) or basal diet plus addition of acetic acid. The dose level was raised every 10-30 days from approximately 155 mg/kg/day to 380-450 mg/kg/day after 60 days. The pigs were weighed during the study and urine samples were taken at intervals to determine ammonia content. At the end of the study blood samples were taken for pH measurement.

GLP compliance:

no

Limit test:

no

Species:

pig

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Not reported - all from same litter
- Age at study initiation: about 100 days old
- Weight at study initiation: 50-60 pounds
- Diet: fed basal diet twice daily. The amount given was based on the smallest amount a group had eaten previously - all groups received the same amount. During the last 30 day period feeding was unresticted basal diet only.
- Water: from drinking troughs allowed ad libitum.
- Acclimation period: All pigs were fed basal diet only for the first 30 days.

ENVIRONMENTAL CONDITIONS
- No details reported

IN-LIFE DATES: From: 4 October 1916 To: April/May 1917

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- The test substance was added to the diet slurry twice daily in the form of up to 500 mL of a 1N aqueous solution

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 months

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
approximately 155 to 450 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

2 pigs (sex not reported)

Control animals:

yes, plain diet

Details on study design:

Following a 30 day period on basal diet, the test group received acetic acid daily, in the diet, starting at a dose of approximately 155 mg/kg/day. Doses were increased every 10 or 30 days until a maximum of 380-450 mg/kg/day was reached after 60 days. Pigs were then maintained on this dose for 3 months, except during period 5 when lactic acid was fed instead of acetic acid (Table 1).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: no data

DETAILED CLINICAL OBSERVATIONS: no data

BODY WEIGHT: Yes
- Time schedule for examinations: At the beginning of each 30 day period and at the end of the experiment

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption determined and mean daily diet consumption calculated : Yes - estimated from pair feeding data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no data

FOOD EFFICIENCY:
- Calculated as amount of feed per 100 lbs live weight

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of urine: taken at the end of the study for pH measurement
URINALYSIS: Yes
- Time schedule for collection of urine: taken in tne morning at intervals to determine ammonia content

Sacrifice and pathology:

GROSS PATHOLOGY: no data
HISTOPATHOLOGY: no data

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Sex:

not specified

Basis for effect level:

other: no adverse effects

Critical effects observed:

not specified

Table 2: Body weight data in pigs fed acetic acid in diet

Parameter	Control	Treated
Mean initial body weight (lbs)	58.6	58.3
Mean final body weight (lbs)	327.4	320.7
Mean daily body weight gain (lbs)	1.28	1.25
Feed/100 lbs body weight gain (g)	439.4	445.8

* 1 lb = 0.45 kg

Conclusions:

There were no mortalities and effects on body weight or acid-base balance in pigs fed acetic acid, at doses of up to 450 mg/kg/day, for approximately 6 months.

Executive summary:

Young pigs (from a single litter) were divided into groups of 2 and fed basal diet (control group) or basal diet plus addition of acetic acid. The dose level was raised every 10-30 days from approximately 155 mg/kg/day to 380-450 mg/kg/day after 60 days. The pigs were weighed during the study and urine samples were taken at intervals to determine ammonia content. At the end of the study blood samples were taken for pH measurement.

There were no mortalities and no effects on body weight or acid-base balance in pigs fed acetic acid, at doses of up to 450 mg/kg/day, for approximately 6 months.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

290 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There are three sufficiently reliable studies available. The chosen NOAEL respresents the worst-case, obtained from the most recent test, so the underestimation of the actual hazard is unlikely and the database is of high quality.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: inhalation

Type of information:

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

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

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

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

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.

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

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

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

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

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:

other: no data

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 300 mg/m³

Study duration:

chronic

Species:

mouse

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

Mode of Action Analysis / Human Relevance Framework

Based on the lack of any observed adverse effects in the relevant study parameters and the absence of any other test item related effects, it is rather impossible to hypothesize a concrete mode of action.

There were no treatment-related deaths. No toxicologically significant clinical observations were detected. There were no toxicologically significant effects in body weight development. No adverse effect on food consumption or food efficiency nor water consumption was detected in treated animals.

There were no toxicologically significant effects detected in the haematological or blood or urine chemical parameters examined. No toxicologically significant macroscopic abnormalities were detected. There were no toxicologically significant effects detected in the organ weights measured, and no treatment related microscopic abnormalities detected, which could be confirmed by clinical parameters.

Summarizing, the oral administration of the read-across substance acetic acid to rats by gavage, at dose levels of 290 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 290 mg/kg bw/day, which is the lowest tested value of all studies available. Similar considerations also apply to the other available studies.

No definitive human relevance framework can be described due to the lack of any other effects securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of relevance of the observed effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

According to Regulation 1272/2008, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values which take into account the duration of exposure and the dose/concentration which produced the effect(s). Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations.

Guidance values for classification into category 2 are 10 < C≤100 mg/kw bw/d in a subchronic study, corresponding to≤300 mg/kg in subacute studies. Due to the lack of adverse effects, the NOAEL could not be determined in all available studies. The lowest available NOAEL is 290 mg/kg bw/d, so it can be reasonably expected that a potential LOAEL woud be way above the guidance value of 300 mg/kg. Further, the slight observed adaptive effects do not indicate any consistent organ affection. Hence, no classification according to Regulation 1272/2008 is triggered.