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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
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.

Data source

Reference
Reference Type:
publication
Title:
Antihypertensive effects of acetic acid and vinegar on spontaneously hypertensive rats
Author:
Kondo S, Tayama K, Tsukamoto Y, Ikeda K and Yamori Y
Year:
2001
Bibliographic source:
Biosci. Biotechnol. Biochem Vol 65, (12), pp 2690-2694

Materials and methods

Test guideline
Qualifier:
no guideline followed
Deviations:
not applicable
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetic acid
EC Number:
200-580-7
EC Name:
Acetic acid
Cas Number:
64-19-7
Molecular formula:
C2H4O2
IUPAC Name:
acetic acid
Details on test material:
- Name of test material (as cited in study report): acetic acid
- Physical state: liquid
- Composition of test material, percentage of components: 46.2 g/L acetic acid

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.