Ammonium acetate

Endpoint summary

Description of key information

Repeated dose toxicity: oral:

Weight of evidence: Read-across approach from experimental results from non-standard studies on rats with Sodium Acetate and Citric acid, sodium salt and experimental results on rats treated with Ammonium Acetate. All these studies showed no signs of toxicity, even though in some studies, the limit dose was exceeded.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: No data on GLP and no guideline was followed. Furthermore, only males were exposed to the substance, the administered doses were really low doses, a small number of animals was used and no separate untreated controls are available for comparison.

Principles of method if other than guideline:

Male rats were chronically treated via drinking water with 50 or 500 ppm (0.005 or 0.05 mg/kg bw/day) sodium acetate from weaning. Behavioral testing on a fixed-ration (FR) schedule of reinforcement began at 55 days of age.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

8 months

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
50 and 500 ppm (0.005 and 0.05 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

Six male rats per dose

Control animals:

no

Observations and examinations performed and frequency:

Body weights were monitored weekly until behavioral testing began, at which point weights were obtained daily.

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):

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):

no effects observed

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 0.05 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on no effects observed at the highest dose tested.

Key result

Critical effects observed:

not specified

No effects were mentioned on survival, reinforcement behaviour or body weight gain.

Conclusions:

No effects were mentioned on survival, reinforcement behaviour or body weight gain.

Executive summary:

Male rats were chronically treated via drinking water with 50 or 500 ppm (0.005 or 0.05 mg/kg bw/day) sodium acetate from weaning. Behavioral testing on a fixed-ration (FR) schedule of reinforcement began at 55 days of age. No effects were mentioned on survival, reinforcement behaviour or body weight gain.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP and no guideline was followed. Furthermore, only males were exposed to the substance, a small number of animals was used, no separate untreated controls are available for comparison and only the survival and growth were studied.

Principles of method if other than guideline:

Male rats of the Wistar strain were used. They came 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. After being weaned at 25 days of age, they were divided at about 28 days of age into experimental groups. Since variation in growth response on the vitamin B12- deficient ration occurred between litters due to such factors as the amount of stored vitamin derived from the mother, one member of each litter was assigned randomly to each group and then started on the experimental rations. Weight gain averages are based on groups of littermates in which both members of the litter survived the experimental period. They were fed ad libitum a 25% protein, vitamin B12-deficient ration either as such or modified by the addition of fatty acids (sodium acetate) for four weeks. The body weights were determined.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Four weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
3.58 % (approximately 3600 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

13 male rats per dose

Control animals:

no

Observations and examinations performed and frequency:

Body weights

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):

not specified

Food efficiency:

not examined

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on no effects observed at the only dose tested

Key result

Critical effects observed:

not specified

No effects on growth or survival were observed at ca. 3600 mg/kg bw/day (the only dose tested).

Conclusions:

No effects on growth or survival were observed at ca. 3600 mg/kg bw/day (the only dose tested).

Executive summary:

Male rats of the Wistar strain were used. They came 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. After being weaned at 25 days of age, they were divided at about 28 days of age into experimental groups. Since variation in growth response on the vitamin B12- deficient ration occurred between litters due to such factors as the amount of stored vitamin derived from the mother, one member of each litter was assigned randomly to each group and then started on the experimental rations. Weight gain averages are based on groups of littermates in which both members of the litter survived the experimental period. They were fed ad libitum a 25% protein, vitamin B12-deficient ration either as such or modified by the addition of fatty acids (sodium acetate) for four weeks. The body weights were determined. No effects on growth or survival were observed at ca. 3600 mg/kg bw/day (the only dose tested).

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: No GLP and no guideline was followed. Furthermore, only males were exposed to the substance, a small number of animals was used and only the body weights and several indices of thyroid function were studied.

Principles of method if other than guideline:

Male Long-Evans rats were randomly divided into two groups which received 0 or 21 mg/kg bw/day sodium acetate in the diet. The investigation was terminated 3 months later and several indices of thyroid function examined.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

3 months

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0 and 21 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

Control: 10 males
Treatment group: 10 males

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

Body weights and several indices of thyroid function

Key result

Dose descriptor:

NOAEL

Effect level:

>= 21 other: mg/kg bw/day (estimated from the food intake)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on no evidence of clearly adverse effects at the only dose tested

Key result

Critical effects observed:

not specified

Indications of altered thyroid function and decreased growth were reported at the only dose tested. However, only males were exposed to the substance, a small number of animals was used and only the body weights and several indices of thyroid function were studied. The reported effects cannot be considered as being clearly adverse. The study is considered to be of limited use in evaluating the toxicity of the substance.

Conclusions:

Indications of altered thyroid function and decreased growth were reported at the only dose tested. However, only males were exposed to the substance, a small number of animals was used and only the body weights and several indices of thyroid function were studied. The reported effects cannot be considered as being clearly adverse. The study is considered to be of limited use in evaluating the toxicity of the substance.

Executive summary:

Male Long-Evans rats were randomly divided into two groups which received 0 or 21 mg/kg bw/day sodium acetate in the diet. The investigation was terminated 3 months later and several indices of thyroid function examined.

Dietary ingestion of sodium acetate for 3 months caused a significant reduction in body weights of male Long-Evans rats. While thyroid gland weights of sodium acetate treated rats were increased significantly above the glands of control rats, thyroid weight related to body weight was almost double that observed in control rats. Moreover, thyroidal 131I uptake was also markedly increased in treated rats but the circulating T4 levels were not significantly different from the serum T4 levels observed in the control group. The enhancement in thyrotropic activity evidenced by the elevated serum TSH levels in rats ingesting sodium acetate was responsible for the increases in thyroid weight and thyroid uptake of radioiodine. Indications of altered thyroid function and decreased growth were reported at the only dose tested. However, only males were exposed to the substance, a small number of animals was used and only the body weights and several indices of thyroid function were studied. The reported effects cannot be considered as being clearly adverse. The study is considered to be of limited used in evaluating the toxicity of the substance.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: No data on GLP and no guideline was followed. Furthermore, only males were exposed to the substance, the administered doses were really low doses, a small number of animals was used and no separate untreated controls are available for comparison.

Principles of method if other than guideline:

Complex maze learning was investigated in male adult rats using a latent learning task. The adult subjects were exposed to sodium acetate in drinking water for 112 days beginning at weaning (day 21 postpartum). Training for the latent learning task began on day 143 for the young adults.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

112 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
100 ppm (approximately 0.01 mg/kg bw/day)
Basis:
nominal in water

No. of animals per sex per dose:

8 male rats per dose

Control animals:

no

Observations and examinations performed and frequency:

Cognitive function

Key result

Dose descriptor:

NOAEL

Effect level:

>= 0.01 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on no effects observed at the only dose tested

Key result

Critical effects observed:

not specified

No evidence of impairment of simple task performance was observed.

Conclusions:

No evidence of impairment of simple task performance was observed. The study is considered to be of limited use in evaluating the toxicity of the substance.

Executive summary:

Complex maze learning was investigated in male adult rats using a latent learning task. The adult subjects were exposed to sodium acetate in drinking water for 112 days beginning at weaning (day 21 postpartum). Training for the latent learning task began on day 143 for the young adults. No evidence of impairment of simple task performance was observed. The study is considered to be of limited use in evaluating the toxicity of the substance.

Reference 5

Endpoint:

repeated dose toxicity: oral

Remarks:

other: read-across from a chronic study with an analogue

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The analogue Sodium Acetate which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the repeated dose toxicity endpoint.

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
See attached reporting format.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from published experimental data from a non-standard study on the analogue Sodium acetate.

GLP compliance:

not specified

Limit test:

no

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):

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):

no effects observed

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

Details on results:

Based on the experimental results obtained with the analogue Sodium acetate (NOAEL >= 0.05 mg/kg bw/day in male Long-Evans rats treated daily by drinking water for 8 months), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.047 mg/kg bw/day.

The analogue Sodium acetate which shares the same functional group with Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:
- a low log Pow value, which is -3.72 for Sodium Acetate and -2.79 for Ammonium acetate,
- a high water solubility, which is 1.25 g/mL at 25 ºC for Sodium acetate and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights, which are 82.03 for Sodium acetate and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 0.047 other: mg/kg bw/day (estimated)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.047 mg/kg bw/day.

Conclusions:

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.047 mg/kg bw/day.

Executive summary:

Based on the experimental results obtained (reported under the endpoint record 07.05.01_01 NaAc) with the analogue Sodium acetate (NOAEL >= 0.05 mg/kg bw/day in male Long-Evans rats treated daily by drinking water for 8 months), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.047 mg/kg bw/day.

Reference 6

Endpoint:

repeated dose toxicity: oral

Remarks:

other: read-across from a subacute study with an analogue

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The analogue Sodium Acetate which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the repeated dose toxicity endpoint.

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
See attached reporting format.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from published experimental data from a non-standard study on the analogue Sodium acetate.

GLP compliance:

no

Limit test:

yes

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):

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

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

Details on results:

Based on the experimental results obtained with the analogue Sodium acetate (NOAEL >= 3600 mg/kg bw/day in male Wistar rats treated daily by feed for 4 weeks), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 3382.76 mg/kg bw/day.

The analogue Sodium acetate which shares the same functional group with Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:
- a low log Pow value, which is -3.72 for Sodium Acetate and -2.79 for Ammonium acetate,
- a high water solubility, which is 1.25 g/mL at 25 ºC for Sodium acetate and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights, which are 82.0 for Sodium acetate and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 382.76 other: mg/kg bw/day (estimated)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 3382.76 mg/kg bw/day.

Conclusions:

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 3382.76 mg/kg bw/day.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.05.01_02 NaAc) obtained with the analogue Sodium acetate (NOAEL >= 3600 mg/kg bw/day in male Wistar rats treated daily by feed for 4 weeks), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 3382.76 mg/kg bw/day.

Reference 7

Endpoint:

repeated dose toxicity: oral

Remarks:

other: read-across from a subchronic study with an analogue

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The analogue Sodium Acetate which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the repeated dose toxicity endpoint.

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
See attached reporting format.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from published experimental data from a non-standard study on the analogue Sodium acetate.

GLP compliance:

no

Limit test:

no

Details on results:

Based on the experimental results obtained with the analogue Sodium acetate (NOAEL >= 21 mg/kg bw/day in male Long-Evans rats treated daily by feed for 3 months), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 19.73 mg/kg bw/day.

The analogue Sodium acetate which shares the same functional group with Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:
- a low log Pow value, which is -3.72 for Sodium Acetate and -2.79 for Ammonium acetate,
- a high water solubility, which is 1.25 g/mL at 25 ºC for Sodium acetate and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights, which are 82.0 for Sodium acetate and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 19.73 other: mg/kg bw/day (estimated)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 19.73 mg/kg bw/day.

Conclusions:

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 19.73 mg/kg bw/day.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.05.01_03 NaAc) obtained with the analogue Sodium acetate (NOAEL >= 21 mg/kg bw/day in male Long-Evans rats treated daily by feed for 3 months), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 19.73 mg/kg bw/day.

Reference 8

Endpoint:

repeated dose toxicity: oral

Remarks:

other: read-across from a subchronic study with an analogue

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The analogue Sodium Acetate which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the repeated dose toxicity endpoint.

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
See attached reporting format.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from published experimental data from a non-standard study on the analogue Sodium acetate.

GLP compliance:

not specified

Limit test:

no

Details on results:

Based on the experimental results obtained with the analogue Sodium acetate (NOAEL >= 0.01 mg/kg bw/day in male Wistar rats treated daily by drinking water for 112 days), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.0094 mg/kg bw/day.

The analogue Sodium acetate which shares the same functional group with Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:
- a low log Pow value, which is -3.72 for Sodium Acetate and -2.79 for Ammonium acetate,
- a high water solubility, which is 1.25 g/mL at 25 ºC for Sodium acetate and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights, which are 82.0 for Sodium acetate and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 0.009 other: mg/kg bw/day (estimated)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.0094 mg/kg bw/day.

Conclusions:

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.0094 mg/kg bw/day.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.05.01_04 NaAc) obtained with the analogue Sodium acetate (NOAEL >= 0.01 mg/kg bw/day in male Wistar rats treated daily by drinking water for 112 days), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.0094 mg/kg bw/day.

Reference 9

Endpoint:

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-standard method and no GLP.

Principles of method if other than guideline:

Two generations of rats were fed 0.1% citric acid, sodium salt in the diet.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet (e.g. ad libitum): Basal diet: Casein 6.0 %, Dried full milk 10.0 %, Whole wheat flour 34.5 %, Potato flour 33.0 %, Peanut oil 6.5 %, Cod liver oil 0.5 %, Linseed oil 0.5 %, Brewer's yeast 5.0 %, Ca2(PO4)2 1.9 %, Na2HPO4 . 2H20 0.8 %, KCl 0.9 %, MgCO3 0.2 %, Na-citrate 0.1 %, various salts 0.1 %.

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Basal diet plus 1.20 % citric acid, sodium salt.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

ca. 1 year

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.1 % (ca. 50 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

No data

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

HAEMATOLOGY: Yes
Blood samples were obtained by amputating the tip of the tail after dilating the tail vein by rubbing the tail with toluene. The erythrocyte number was determined by measuring the extinction of the 20-fold diluted blood at 650 µm.

URINALYSIS: Yes
During the metabolism experiments the animals were kept in metabolism cages and the urine was collected separately from the feces over periods of seven days. The titratable acidity was determined according to Henderson and Palmer. For ammonia and urea the microdiffusion technique was used. Total N was determined with the Kjeldahl technique, and inorganic phosphate by the method of Sumner. Calcium was determined according to Wang. Total base was determined by an ion exchange method. For citric acid the method of Weil-Malherbe and Bone was used.

FECES:
The feces were dried overnight at 105 ºC, weighed and ground. Fecal total phosphorus was determined in a nitric acid-perchloric acid digest by means of the method of Sumner. Calcium was detemined in the same digest with the method of Wang. Total N was determined with the Kjeldahl technique.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
A limited number of tissues were examined microscopically.

Other examinations:

BLOOD
The animals were killed by decapitation and the collected blood was centrifuged immediately. The inorganic phosphorus was determined by the method of Sumner and calcium by means of the procedure of Wang. For total base an ion exchange method was used. The manometric method of Van Slyke was used for determining the total carbon dioxide content of the serum. Chloride was determined with an iodometric method. The method of Bessey et al. was employed for the determination of alkaline phosphatase activity in serum.

TISSUES
Total phosphorus was determined in a nitric acid-perchloric acid digest by means of the method of Sumner. In the same digest Na and K were determined flame photometrically, using blanks and standards containing approximately the same amounts of nitric and perchloric acid as were present in the digests. The alkaline phosphatase activity of the kidney was determined by the method of Bessey et al. in a 2.5 % homogenate.
The tibiae were ashed at 600 ºC after extraction of the lipids with ether ethanol. The ash was dissolved in dilute hydrochloric acid and the phosphorus determined with the method of Sumner and the calcium with Wang's method.

TEETH
The method of Keyes as modified by Jansen et al. was used for the quantitative appraisal of the caries. The degree of dental erosion was also observed. Histological preparations were made of some of the molars by the method of Mann as modified by Dobell.

Statistics:

The statistical methods used in the evaluation of the results have been described in detail. The bilateral tail probability (p), i.e. the probability that the difference found be accidental, was calculated in each instance. The difference was considered significant, whenever p was smaller than 0.05.

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):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

No harmul effects were produced by the acid diet in two successive generations of animals maintained on this diet for a considerable part of the life span, as shown by an extensive study of growth, reproduction, the blood picture, the gross and microscopical appareance of the organs, the mineral and nitrogen metabolism and the tissue composition.
The normal physiological processes for the removal of ingested acid were found to be sufficient even after a continuous intake in two successive generations.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

No adverse effects were found. The NOAEL was equal or greater than 50 mg/kg bw/day.

Conclusions:

No adverse effects were found. The NOAEL was equal or greater than 50 mg/kg bw/day.

Executive summary:

Albino rats were maintained on diets to which citric acid, sodium salt, was added in a quantity that represented, on the basis of body weight, the maximum daily intake of acid that would be possile if the entire daily caloric requirements were obtained solely from the sugar contained in soft drinks. The exposure lasted for ca. 1 year.

No harmul effects were produced by the acid diet in two successive generations of animals maintained on this diet for a considerable part of the life span, as shown by an extensive study of growth, reproduction, the blood picture, the gross and microscopical appareance of the organs, the mineral and nitrogen metabolism and the tissue composition.

The normal physiological processes for the removal of ingested acid were found to be sufficient even after a continuous intake in two successive generations.

The NOAEL was equal or greater than 50 mg/kg bw/day.

Reference 10

Endpoint:

repeated dose toxicity: oral

Remarks:

other: read-across from a chronic study with an analogue

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: The analogue Citric acid, sodium salt which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the repeated dose toxicity endpoint.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See attached reporting format.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from published experimental data from a non-standard study on the analogue Citric acid, sodium salt.

GLP compliance:

no

Limit test:

no

Details on results:

Based on the experimental results obtained with the analogue Citric acid, sodium salt (NOAEL >= 50 mg/kg bw/day in rats treated daily by feed for ca. 1 year), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 54 mg/kg bw/day.

The analogue Citric acid, sodium salt, which shares the same functional group with Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:
- a low log Pow value which is -0.28 for Citric acid, sodium salt, and -2.79 for Ammonium acetate,
- a high water solubility which is 1 g/mL for Citric acid, sodium salt, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 214.11 for Citric acid, sodium salt, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 54 other: mg/kg bw/day (estimated)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

Both chemicals are grouped together by US EPA category group Carboxylic Food Acids and Salts Category.

As indicated in the European Chemical Agency Practical Guide 6 “How to report read –across and categories”, the structural grouping was realized using “OECD QSAR APPLICATION TOOL BOX” version 1.1.0.Presented results show that both substances have common (eco)toxicological behavior (attachment).

Table 1. Data Matrix, Analogue Approach.

CAS Number		Source chemical 994-36-5	Target chemical 631-61-8
CHEMICAL NAME		Citric acid, sodium salt	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		No data	Experimental data: 114 ºC
Boiling Point		No data	Estimated data: 312.76 ºC
Density		No data	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Negligible	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Estimated data: -0.28	Estimated data: -2.79
Water solubility		Estimated data: 1000 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		No data	Experimental results on Ammonium Acetate, read-across from experimental data on Sodium Acetate and read-across from estimated data on Ammonia and Acetic Acid, based on functional group:   Readily biodegradable
ENVIRONMENTAL TOXICITY
Acute Toxicity to Fish		No data	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		No data	Read-across from experimental data on analogues Sodium Acetate, Potassium Acetate and Ammonia, based on molecular weights:   EC50 = 108.81 - 939.66 mg/L
Toxicity to Aquatic Plants		No data	Read-across from experimental data on analogues Acetic Acid, Potassium Acetate and Ammonium Sulphate, based on molecular weights: (72 h) EC50 > 392.70 mg/L; (72 h) NOEC = 392.70 mg/L.
MAMMALIAN TOXICITY
Acute Toxicity: Oral		Experimental data: LD 50 = 7100 mg/kg bw (mice)	Weight of evidence: Read-across from experimental data on Potassium Acetate and Ammonium Sulphate, based on molecular weights: LD50 = 2333.28-3546.59 mg/kg bw
Acute Toxicity: Inhalation		No data	No data
Acute Toxicity: Dermal		No data	Weight of evidence: Read-across from experimental data on Fumaric Acid and Ammonium Sulphate, based on molecular weights: LD50 = 2333.28-26556.42 mg/kg bw
Skin Irritation/Corrosion		No data	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate, Ammonium Sulphate, and Ammonium Stearate, based on functional group: The substance Ammonium Acetate is considered as not irritating for eyes.
Eye Irritation/Corrosion		No data	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Stearate, based on functional group: The substance Ammonium Acetate is considered as not irritating for eyes.
Skin sensitisation		No data	Weight of evidence:   Read-across approach from experimental results on Citric Acid, Glycolic Acid, Sodium Glycolate, Lactic Acid, Ammonium Lactate, and Triacetin, based on functional group:   All this substances were not sensitising for human and guinea pigs. Based on these results, Ammonium acetate is considered to be not sensitizing.
Repeated Dose Toxicity		Repeated dose toxicity: oral: Experimental results:   In a 1 year study with rats daily treated by feed, theNOAEL was ca. 50 mg/kg bw/day(based on no effects observed at the only dose tested).	Repeated dose toxicity: oral: Weight of evidence: Experimental results:   Repeated dose toxicity: oral: 90 days withfemale Wistar rats. The NOAEL was 3150.4 mg/kg bw/day . Repeated dose toxicity: oral: 15 days study with female Wistar rats. The NOAEL was 3102.2 mg/kg bw/day . Read-across from the analogue Sodium Acetate, based on molecular weights:   The NOAEL >= 0.047 mg/kg bw/day, in male rats chronically treated for 8 months via drinking water. The NOAEL >= 3382.76 mg/kg bw/day, in male Wistar rats daily treated for 4 weeks by feed. The NOAEL >= 19.73 mg/kg bw/day, in male Long-Evans rats treated for 3 months in the diet. The NOAEL >= 0.0094 mg/kg bw/day, in male Wistar rats treated by drinking water for 112 days.    Read-across from the analogue Citric acid, sodium salt, based on molecular weights:   The NOAEL >= 54 mg/kg bw/day, in albino rats treated for ca. 1 year.
Genetic Toxicity in vitro	-         Gene mutation in bacteria	Experimental data:   Citric acid, sodium salt was tested in an assay usingSaccharomyces cerevisiaecells in the presence and absence of metabolic activation; however, insufficient information was provided in the robust summary of this study to adequately evaluate the results.	Weight of evidence:   Read-across from Sodium Acetate (category analogue) based on functional group:   Reverse mutation assay using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with metabolic activation. Resultslead to the conclusion that Ammonium Acetate did not cause point mutations in the microbial systems. Read-across from Acetic Acid, based on functional group:   Ammonium Acetate is considered to be not mutagenic on S.typhimurium TA 98, TA 100, TA 1535, TA 97, and/or TA 1537, with and without metabolic activation. Read-across from experimental data on Ammonia, anhydrous, based on functional group: Ammonium acetate is considered to be not mutagenic on Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538, and Escherichia coli WP2uvrA, with and without metabolic activation.   Read-across from experimental data on Ammonia, aqueous solution, based on functional group: Ammonium acetate is considered not mutagenic on E. coli Sd-4-73, without metabolic activation.
	-         Mammalian gene mutation	No data	Weight of evidence: Read-across from the analogue Acetic anhydride, based on functional group: Ammonium acetate is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation. Read-across from the analogue Phenoxy acetic acid, based on functional group: Ammonium acetate is considered to be not mutagenic on Chinese hamster ovary cells, with and without metabolic activation.   Estimated data from Danish (Q)SAR Database: Ammonium acetate was not mutagenic in mammalian cell gene mutation assays on mouse lymphoma L5178Y cells nor on Chinese hamster ovary cells.
	Chromosomal aberration	No data	Weight of evidence: Read-across from Sodium Acetate (category analogue) based on functional group:   In an in vitro chromosomal aberration assay with a Chinese hamster fibroblast cell line, CHL, without metabolic activation systems, it is concluded that Ammonium acetate did not induce chromosomal aberrations(including gaps). Read-across from Acetic Acid, based on functional group:   Ammonium Acetate is considered as not clastogenic on Chinese hamster Ovary (CHO) cells, without metabolic activation. Read-across from Ammonium Sulfate, based on functional group: Ammonium Acetate is not considered mutagenic on Chinese Hamster Ovary cells, in the absence of a metabolic activation system.
Genetic Toxicity in vivo		No data	Key studies: Read-across from Sodium Acetate (category analogue) based on functional group:   The Testicular DNA-synthesis inhibition test (DSI test) on male mice provides evidence that Ammonium acetate is not genotoxic in animals (basis of the method: measuring 3H-thymidine incorporation). Test substance did not inhibit DNA replication in this assay.
Carcinogenicity		No data	No data
Reproductive Toxicity		TOXICITY TO REPRODUCTION: Experimental results:   In a 1 year study with rats daily treated by feed, theNOAEL was ca. 50 mg/kg bw/day(based on no effects observed at the only dose tested). DEVELOPMENTAL TOXICITY / TERATOGENICITY:   No data	TOXICITY TO REPRODUCTION: Weight of evidence: Read-across from the analogue Citric Acid, based on molecular weights: A study on rats and mice daily treated by feed before, during, and after mating. For Ammonium Acetate, the NOAEL is calculated to be equal or greater than 3009.37 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young). A fertility test on female rats daily treated by feed for several months. For Ammonium Acetate, the NOAEL is calculated to be 722.25 mg/kg bw/day, and LOAEL greater than 722.25 mg/kg bw/day for reproductive effects. Read-across from the analogue Citric Acid, sodium salt, based on molecular weights: A fertility study on female rats daily treated by feed for several months. For Ammonium Acetate, the NOAEL is calculated to be 54.0 mg/kg bw/day, and LOAEL greater than 54.0 mg/kg bw/day for reproductive effects. Read-across from the analogue Ammonium sulfate, based on molecular weights: A study on male and female rats exposed for 13 weeks to diets with Ammonium Sulfate. For Ammonium Acetate, the NOAEL is calculated to be 1033.64 mg/kg bw/day for males, and 2304.12 mg/kg bw/day for females.   DEVELOPMENTAL TOXICITY / TERATOGENICITY: Weight of evidence: Experimental results: A study on female rats fed an ammonium-containing diet starting on day 1 of pregnancy until weaning (at posnatal day on 21). After weaning, pups were either fed a normal diet, with no ammonium acetate added, or continued on ammonium until sacrifice. The NOAEL for developmental toxicity was 4293 mg/kg bw/day . Read-across from the analogue Sodium Acetate, based on molecular weights: Pregnant CD-1 mice were treated by oral gavage with Sodium Acetate on days 8-12 of gestation. For Ammonium Acetate, theNOAEL is calculated to be939.66 mg/kg bw/day (based on maternal toxicity: mortality, pregnancy and resorption; and on neonatal effects: mortality and body weight). Read-across from the analogue Citric Acid, based on molecular weights: A study on rats and mice daily treated by feed before, during, and after mating. For Ammonium Acetate, the NOAEL is calculated to be equal or greater than 3009.37 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young). Read-across from the analogue substance Calcium Formate, based on molecular weights: A three-generation drinking water study was performed. For Ammonium Acetate, the NOAEL is calculated to be equal or higher than 236.96 mg/kg bw/day. Read-across from Acetic Acid, based on molecular weights: A one-generation study was performed on female mice, rats and rabbits with Acetic Acid. The read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 2055.47 mg/kg bw/day for maternal and developmental toxicity in mice, rats, and rabbits.

Conclusions:

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 54 mg/kg bw/day.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.05.01_09 Citric Acid sodium salt) obtained with the analogue Citric acid, sodium salt (NOAEL >= 50 mg/kg bw/day in rats treated daily by feed for ca. 1 year), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 54 mg/kg bw/day.

Reference 11

Endpoint:

repeated dose toxicity: oral

Remarks:

other: subacute + subchronic

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No specific test guideline was reported; however, a scientifically defensible approach was used to conduct the study.No data on GLP.

Principles of method if other than guideline:

The effect of a recent hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, to female Wistar rats has been studied. The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. Fifty female rats were divided into ten groups (five control and five experimental groups), each one including five animals.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 200-250 g
- Diet and Water (e.g. ad libitum): Control rats were fed ad libitum with a standard diet (UAR A04, Panlab, Spain) (17% protein); the experimental animals were fed ad libitum with a high ammonia diet and received ammonium acetate in the water supply (5mM).

Route of administration:

other: feed + drinking water

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
The high ammonia diet was prepared by mixing standard diet with ammonium acetate (20% m/wt) as described by Azorin et al.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The ammonia concentrations in the standard and high ammonia diets were determined by means of the Lucke and Giedel's method and were 0.024% and 3.377% respectively (g NH4/100 g food).

Duration of treatment / exposure:

3, 7, 15, 45, or 90 days.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
20% ammonium acetate in the diet and 5 mM ammonium acetate in the drinking water
Basis:
other: nominal in diet and nominal in water

No. of animals per sex per dose:

Fifty female rats were divided into ten groups (five control and five experimental groups), each one including five animals.

Control animals:

yes, concurrent no treatment

Positive control:

No data

Observations and examinations performed and frequency:

Blood samples for ammonia determination were collected at the moment of decapitation; plasma was separated immediately and stored at - 70 ºC.
Blood ammonia level was determined with an Ammonia Kit.

Sacrifice and pathology:

Each group was sacrificed at different times: 3, 7, 15, 45, or 90 days.
Rats were killed by decapitation and the spinal cord was immediately removed as described by Meikle and Martin and frozen at - 80 ºC in sodium dodecyl sutfate (SDS) extraction buffer (0.15% SDS, 0.05 M phosphate buffer, 0.02% EDTA and 0.9% NaCl; pH:8).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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

Details on results:

No observable differences were noted in either the behaviour or the water consumption between control and high ammonia diet rats; however, significative differences in weight were observed after the third day.
At dose of 3150.4 mg/kg bw/day: 10 % reduction in final body weight at 3 or 7 days
15 % reduction in final body weight at 90 days

Blood ammonia analysis demonstrated a significative difference between high ammonia diet and control animals at 7th day. No significant differences between experimental and control animals were observed at the other times studied.

Key result

Dose descriptor:

LOAEL

Effect level:

3 150.4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

not specified

The NOAEL was 22 mg/kg bw/day and the LOAEL was 3150.4 mg/kg bw/day .

Conclusions:

The NOAEL was 22 mg/kg bw/day and the LOAEL was 3150.4 mg/kg bw/day .

Executive summary:

The effect of a recent hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, to female Wistar rats has been studied. The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. Fifty female rats were divided into ten groups (five control and five experimental groups), each one including five animals.

Blood ammonia analysis demonstrated a significative difference between high ammonia diet and control animals at 7th day. No significant differences between experimental and control animals were observed at the other times studied.

No observable differences were noted in either the behaviour or the water consumption between control and high ammonia diet rats; however, significative differences in weight were observed after the third day. The LOAEL was 3150.4 mg/kg bw/day.

Reference 12

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: No specific test guideline was reported; however, a scientifically defensible approach was used to conduct the study. No data on GLP.

Principles of method if other than guideline:

Female Wistar rats were fed a high ammonia diet prepared by mixing a standard diet with ammonium acetate (20% w/w); in addition, 5 mM of ammonium acetate was added to their water supply. Control rats were fed with a standard diet. The animals were sacrificed at 3, 7 and 15 days of ammonia ingestion. Thirty female rats were divided into six groups (three control and three experimental groups), each one including five animals.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 220-250 g
- Diet and Water (e.g. ad libitum): Control rats were fed ad libitum with a standard diet (UAR A04, Panlab, Spain) (17% protein); the experimental animals were fed ad libitum with a high ammonia diet and received ammonium acetate in the water supply (5mM).

Route of administration:

other: feed + drinking water

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
The high ammonia diet was prepared by mixing standard diet with ammonium acetate (20% m/wt) as described by Azorin et al.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

3, 7, or 15 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
20% ammonium acetate in the diet and 5 mM ammonium acetate in the drinking water
Basis:
other: nominal in diet and nominal in water

No. of animals per sex per dose:

Thirty female rats were divided into six groups (three control and three experimental groups), each one including five animals.

Control animals:

yes, concurrent no treatment

Positive control:

No data

Observations and examinations performed and frequency:

Blood samples for ammonia determinations were collected at the moment of decapitation; plasma was separated immediately and stored at - 70 ºC.
Blood ammonia level was determined with an Ammonia Kit.

Sacrifice and pathology:

The animals were killed by decapitation at 3, 7 and 15 days after starting treatment.
The brains were removed and the frontoparietal cortex and hippocampus were rapidly dissected.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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

Details on results:

Significant differences in weight were observed by the third day on the ammonia diet and were maintained for up to 15 days. Ammonia levels in blood increased from 0.13 mM in control rats to 0.4 mM in rats fed the high ammonia diet at 7 days of starting this diet.
Rats had a decreased number of available somatostatin receptors in the frontoparietal cortex and hippocampus and lower weigh increase.

Key result

Dose descriptor:

NOAEL

Effect level:

3 102.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

not specified

The NOAEL 3102.2 mg/kg bw/day.

Conclusions:

The NOAEL for Ammonium acetate was 3102.2 mg/kg bw/day

Executive summary:

Female Wistar rats were fed a high ammonia diet prepared by mixing a standard diet with ammonium acetate (20% w/w); in addition, 5 mM of ammonium acetate was added to their water supply. Control rats were fed with a standard diet. The animals were sacrificed at 3, 7 and 15 days of ammonia ingestion. Thirty female rats were divided into six groups (three control and three experimental groups), each one including five animals.

Significant differences in weight were observed by the third day on the ammonia diet and were maintained for up to 15 days.

Ammonia levels in blood had increased ca. 3-fold at 7 days of ammonia ingestion. Rats with high NH4+ intake from administration of 20% ammonium acetate in the diet and 5 mM ammonium acetate in the water for up to 15 days had a decreased number of available somatostatin receptors in the frontoparietal cortex and hippocampus.

The NOAEL was 3102.2 mg/kg bw/day.

Reference 13

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: Test method was similar to OECD guideline. No data on GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Fischer 344 rats (10 rats/sex/dose) were exposed during 13 weeks to diets containing 0, 0.38, 0.75, 1.5 or 3 % ammonium sulfate (corresponding to 0, 222, 441, 886, 1792 mg/kg bw/day in males and to 0, 239, 484, 961, 1975 mg/kg bw/day in females).

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: SPF F344/DuCri rats from Charles River Nippon.
- Age at study initiation: (P) 5 week old
- Diet (e.g. ad libitum): Animals were maintained on CRF-1 powder diet.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ºC
- Humidity (%): 55 ± 5 %
- Air changes: 18 air changes/day
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

Route of administration:

oral: feed

Details on route of administration:

The animals were fed CRF-1 powder diet containing concentrations of 0, 0.38, 0.75, 1.5, and 3.0% of
ammonium sulfate.

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continously

Dose / conc.:

0.38 other: %

Remarks:

(222 mg/kg bw/day in males and 239 mg/kg bw/day in females)

Dose / conc.:

0.75 other: %

Remarks:

(441 mg/kg bw/day in males and 484 mg/kg bw/day in females)

Dose / conc.:

1.5 other: %

Remarks:

(886 mg/kg bw/day in males and 961 mg/kg bw/day in females)

Dose / conc.:

3 other: %

Remarks:

(1792 mg/kg bw/day in males and 1975 mg/kg bw/day in females)

No. of animals per sex per dose:

10 males and 10 females per dose.

Control animals:

yes, concurrent no treatment

Details on study design:

The dose levels were set on the basis of results from a previous 2-week study with a dose level of 5% .

Observations and examinations performed and frequency:

Parental animals: Observations and examinations
CINICAL CHEMISTRY, HEMATOLOGY: The following clinical parameters were determined: total protein (TP), albumin/globulin (A/G), albumin, total cholesterin, BUN, Na, Cl, K, Ca, P, GOT, GPT, alkaline phosphatase. Hematology included red blood cell count (RBC), hemoglobin (Hb), hemato
crit (Hk), mean corpuscular volume (MCV), mean erythrocyte hemoglobin (MCH), mean erythrocyte
hemoglobin concentration (MCHC), platelet count, and leukocyte count.

Postmortem examinations (parental animals)
ORGANS WEIGHED: At necropsy, the following organs were weighed and absolute and relative organ weights determined:
Males: brain, lung, heart, spleen, liver, adrenal, kidney, testis;
Females: brain, lung, heart, spleen, liver, adrenal, kidney.

ORGANS EXAMINED HISTOPATHOLOGICALLY:
Males: brain, lung, heart, spleen, liver, adrenal, kidney, testis.
Females: brain, lung, heart, spleen, liver, adrenal, kidney.

Organs were fixed in formalin and hematoxylin-eosin slides were prepared and examined histologically.

Statistics
Bartlett,and Kruskal - wallis tests, and parametric Dunnett and Scheffe tests.

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Description (incidence and severity):

BODY WEIGHTS: at study end final body weights were in males 298, 273, 287, 282 and 284 g for the
0, 0.38, 0.75, 1.5 and 3% groups, respectively. In females, final body weights were 151, 157, 152, 161 and 158 g for the 0, 0.38, 0.75, 1.5 and 3% groups, respectively.

Description (incidence and severity):

FOOD INTAKE (g/rat/day): 14.2, 14.0, 14.3, 14.1, 13.8 in the males of the 0, 0.38, 0.75, 1.5 and 3%
groups, respectively. In females, the values were 9.2, 9.1, 9.3, 9.3 and 8.4 for the 0, 0.38, 0.75, 1.5 and 3% groups, respectively.

Description (incidence and severity):

HEMATOLOGY AND CLINICAL CHEMISTRY PARAMETERS: No biologically significant changes were observed in any of the investigated parameters. Though there were statistical differences in some of the parameters, there was no consistent dose-effect relationship and/or all values were
within the normal ranges of values normally found in the rat strain used in this study. In particular, the re were no signs indicative of a metabolic acidosis.

Description (incidence and severity):

ORGAN WEIGHTS: No significant changes in absolute or relative organ weights were observed for brain, lung, heart, spleen, liver, adrenals, kidney and testis weights. Increases (<15%) in the relative and absolute kidney weights in high dose male and females, and in liver weight in high dose females
(+11%), were not accompanied by any functional (clinical parameters) or histopathological changes, and were therefore not considered as adverse effects by the authors.

Description (incidence and severity):

HISTOPATHOLOGICAL EXAMINATIONS: No significant pathological effects were found. In the
3% male group myofibrosis cordis, basophilic kidney tubulus as well as spleenic melanosis were observed; in the female group basophilic kidney tubulus as well as splenic melanosis were observed.
However the rate of occurrence was similar to controls.

Key result

Dose descriptor:

NOAEL

Effect level:

886 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

NOAEL

Effect level:

1 975 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

no

Conclusions:

The NOEL was judged to be 886 mg/kg bw/day in males and 1975 mg/kg bw/day in females.

Executive summary:

Fischer 344 rats (10 rats/sex/dose) were exposed during 13 weeks to diets containing 0, 0.38, 0.75,

1.5 or 3 % ammonium sulfate (corresponding to 0, 222, 441, 886, 1792 mg/kg bw/day in males and to

0, 239, 484, 961, 1975 mg/kg bw/day in females).

Diarrhea was observed in males at 3% during the exposure period. Increased kidney weights in

males and females at 3% and increased liver weights in females at 3% were not accompanied

by histopathological changes. The relative testes weight was significantly increased at all doses,

but no histological effects were found. No effect was observed on body weight, food consumption,

hematological and clinical parameters. No effects on the reproductive organs were observed.

Therefore, the NOAEL was 886 mg/kg bw/day for males and 1975 mg/kg bw/day for females.

Reference 14

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:

Justification for type of information:

The analogue Ammonium sulfate which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the repeated dose toxicity endpoint.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from published experimental data on fertility on the analogue Ammonium sulfate.

GLP compliance:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

1 033.64 mg/kg bw/day (nominal)

Sex:

male

Remarks on result:

other: overall effects

Key result

Dose descriptor:

NOAEL

Effect level:

2 304.12 mg/kg bw/day (nominal)

Sex:

female

Remarks on result:

other: overall effects

Key result

Critical effects observed:

no

Conclusions:

The NOAEL with the substance Ammonium acetate is calculated to be 1033.64 mg/kg bw/day for
males, and 2304.12 mg/kg bw/day for females.

Executive summary:

Based on the experimental results (reported under endpoint record 07.05.01_13 Ammonium sulfate) obtained with the analogue Ammonium sulfate on rats daily treated by feed for 13 weeks (NOAEL for overall effects is 886 mg/kg bw/day in males, and 1975 mg/kg bw/day in females), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be 1033.64 mg/kg bw/day for males, and 2304.12 mg/kg bw/day for females.

Endpoint conclusion

Dose descriptor:

NOAEL

3 102.02 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Repeated-dose toxicity: oral:

Experimental results with Ammonium Acetate:

In the publication reported by Bodega et al. (1993), the effect of a hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, to female Wistar rats has been studied. The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. Fifty female rats were divided into ten groups (five control and five experimental groups), each one including five animals.

No observable differences were noted in either the behaviour or the water consumption between control and high ammonia diet rats; however, significative differences in weight were observed after the third day. The LOAEL was 3150.4 mg/kg bw/day.

In the publication reported by Boyano et al. (1996), female Wistar rats were fed a high ammonia diet prepared by mixing a standard diet with ammonium acetate (20% w/w); in addition, 5 mM of ammonium acetate was added to their water supply. The animals were sacrificed at 3, 7 and 15 days of ammonia ingestion. Thirty female rats were divided into six groups (three control and three experimental groups), each one including five animals.

The NOAEL was 3102.2 mg/kg bw/day.

Read-across approach from experimental results from non-standard studies with Sodium Acetate and Citric acid, sodium salt, Ammonium sulfate.

In the publication reported by Cory-Slechta DA (1986), male rats were chronically treated for 8 months via drinking water with 50 or 500 ppm (0.005 or 0.05 mg/kg bw/day) Sodium Acetate from weaning. No effects were mentioned on survival, reinforcement behaviour or body weight gain.

Based on this data and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.047 mg/kg bw/day.

In the second paper, reported by Dryden LP et al. (1971), effects of Sodium Acetate were studied on male Wistar rats daily treated for 4 weeks by feed with ca. 3600 mg/kg bw/day. No effects on growth or survival were observed at this dose.

The read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 3382.76 mg/kg bw/day.

In the third publication, reported by Goldman M (1981), male Long-Evans rats received daily 0 or 21 mg/kg bw/day Sodium Acetate in the diet. The investigation was terminated 3 months later and several indices of thyroid function examined. The reported effects cannot be considered as being clearly adverse. This study is considered to be of limited use in evaluating the toxicity of the substance.

Applying the read-across approach, the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 19.73 mg/kg bw/day.

In the study reported by Massaro EJ et al. (1986), Sodium Acetate was daily administered to male Wistar rats by drinking water for 112 days beginning at weaning (day 21 postpartum). Training for the latent learning task began on day 143 for the young adults. No evidence of impairment of simple task performance was observed. This study is also considered to be of limited use in evaluating the toxicity of the substance. The NOAEL was equal or greater than 0.01 mg/kg bw/day.

The read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 0.0094 mg/kg bw/day.

The next study, reported by Bonting SL et al. (1956) was performed with the analogue substance Citric acid, sodium salt. Albino rats were maintained on diets to which citric acid, sodium salt was added in a quantity that represented, on the basis of body weight, the maximum daily intake of acid that would be possile if the entire daily caloric requirements were obtained solely from the sugar contained in soft drinks. The exposure lasted for ca. 1 year.

No harmul effects were produced by the acid diet in two successive generations of animals maintained on this diet for a considerable part of the life span, as shown by an extensive study of growth, reproduction, the blood picture, the gross and microscopical appareance of the organs, the mineral and nitrogen metabolism and the tissue composition. The NOAEL was equal or greater than 50 mg/kg bw/day (nominal in diet).

Based on these results obtained with the analogue Citric acid, sodium salt and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 54 mg/kg bw/day.

Based on the experimental results obtained with the analogue Ammonium sulfate on rats daily treated by feed for 13 weeks (NOAEL for overall effects is 886 mg/kg bw/day in males, and 1975 mg/kg bw/day in females), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be 1033.64 mg/kg bw/day for males, and 2304.12 mg/kg bw/day for females.