Ammonium acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study meets generally accepted scientific principles, so it is acceptable for assessment. No GLP.

Principles of method if other than guideline:

This study decribes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of the rats. Animals were fed a standard bran/oats dried milk diet. Control groups of female rats were compared to groups of female rats fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

not specified

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet (e.g. ad libitum): Animals were fed a standard bran/oats dried milk diet.

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Animals were treated before, during, and after mating.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
5 % (about 2.5 g/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

6 female rats per group

Control animals:

yes

Positive control:

No data

Parental animals: Observations and examinations:

Number of pregnancies in rats.

Litter observations:

Number of young born, or survival of young in rats.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

In the reproduction studies, no effects were seen on number of pregnancies in rats.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (number of pregnancies)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

No effects were seen on number of young born, or survival of young in rats.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (number of young born, or survival of young animals).

Key result

Reproductive effects observed:

not specified

No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats with ca. 2.5 g/kg bw/day, compared to controls.

Conclusions:

No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats with ca. 2.5 g/kg bw/day, compared to controls.

Executive summary:

This study decribes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of the rats. Animals were fed a standard bran/oats dried milk diet. Control groups of female rats were compared to groups of female rats fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats with ca. 2.5 g/kg bw/day, compared to controls.

Reference 2

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study meets generally accepted scientific principles, so it is acceptable for assessment. No GLP.

Principles of method if other than guideline:

This study describes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of mice. Animals were fed a standard bran/oats dried milk diet. Control groups of female mice were compared to groups of female mice fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

GLP compliance:

no

Limit test:

yes

Species:

mouse

Strain:

not specified

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet (e.g. ad libitum): Animals were fed a standard bran/oats dried milk diet.

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Animals were treated before, during, and after mating.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
5 % (about 2.5 g/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

6 female mice per group

Control animals:

yes

Positive control:

No data

Parental animals: Observations and examinations:

Number of pregnancies in mice.

Litter observations:

Number of young born, or survival of young in mice.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

In the reproduction studies, no effects were seen on number of pregnancies in mice.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (number of pregnancies)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

No effects were seen on number of young born, or survival of young in mice.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (number of young born, or survival of young animals).

Key result

Reproductive effects observed:

not specified

No effects were seen on number of pregnancies, number of young born, or survival of young in treated mice with ca. 2.5 g/kg bw/day, compared to controls.

Conclusions:

No effects were seen on number of pregnancies, number of young born, or survival of young in treated mice with ca. 2.5 g/kg bw/day, compared to controls.

Executive summary:

This study decribes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of mice. Animals were fed a standard bran/oats dried milk diet. Control groups of female mice were compared to groups of female mice fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

No effects were seen on number of pregnancies, number of young born, or survival of young in treated mice with ca. 2.5 g/kg bw/day, compared to controls.

Reference 3

Endpoint:

one-generation reproductive toxicity

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 which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 on fertility and one-generation study on the analogue Citric acid.

GLP compliance:

no

Limit test:

yes

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day (basis for effect: number of pregnancies) in rats daily treated by feed before, during, and after mating), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (number of pregnancies)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in rats (basis for effect: number of young born, or survival of young animals)), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (number of young born, or survival of young animals).

Key result

Reproductive effects observed:

not specified

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,

- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights, which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

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 77-92-9	Target chemical 631-61-8
CHEMICAL NAME		Citric acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 153 ºC	Experimental data: 114 ºC
Boiling Point		Decomposes	Estimated data: 312.76 ºC
Density		Measured data: 1.665 g/cm3 at 20 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Estimated data: 5.6E-09 mm Hg	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -1.72	Estimated data: -2.79
Water solubility		Measured data: 1330 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 1516 mg/L	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 1535 mg/L	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		Experimental data: (72 h) EC 50 = 640 mg/L	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 = 5790 – 7100 mg/kg bw (mice) LD 50 = 11700 mg/kg bw (rats)	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		Experimental data:   A 4-hour skin irritation study in rabbits exposed to a solution containing 60% citric acid caused erythema and edema. This study did not assess a lethal dose value. TheLD50 was not provided.	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		Experimental data:   Application of 500 mg Citric Acidto rabbit skin produced moderate irritation in 24 hr.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Citric Acidtested on rabbit eyes as single drop of 2% to 5% solution in water caused little or no injury. Irrigation for 30 min with 0.5% to 2% solution causes severe injury; the 0.5% solution causes permanent cloudiness of cornea, and the 2% solution causes severe dense opacification.   Application of 750 mg caused severe irritation in the rabbit eye.	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.
Skin sensitisation		Experimental results:   Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.   No allergic reactions were seen when 60 patients with hand eczema, all of whom were involved in handling food, were patch tested (covered contact, probably 24 hr) with 2.5% citric acid in petrolatum.	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 150-day study with male New Zealand White rabbits daily treated by feed, theNOAEL was 1500 mg/kg bw/day(based on no effects observed at the only dose tested).   In a 6-week study with male Sprague-Dawley rats daily treated by feed, theNOAEL was 4800 mg/kg bw/day(based on no effects observed at the highest 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:   In a bacterial reverse mutation assays usingS. typhimurium(TA97, TA98, TA100 and TA104) in the presence and absence of metabolic activation and up to 2000μg/plate, citric acid was not mutagenic.	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		Experimental data:   In a Dominant Lethal assay using male/female rats dosed at 3 g/kg for 5 days, citric acid did not induced germ cell genotoxicity.	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 data: In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young). In a fertility study, female rats were exposed to citric acid in their daily diet for several months. TheNOAEL (reproductive toxicity) was 600 mg/kg bw/day(based on no effects observed at the only dose tested).   DEVELOPMENTAL TOXICITY / TERATOGENICITY:   Experimental data:   In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young).	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 3009.37 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young animals).

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.01_01 Citric acid) obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in rats (basis for effect: number of pregnancies, number of young born, or survival of young animals)), 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 3009.37 mg/kg bw/day for studied effects.

Reference 4

Endpoint:

one-generation reproductive toxicity

Type of information:

migrated 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 which shares the same functional group with Ammonium acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 on fertility and one-generation study on the analogue Citric acid.

GLP compliance:

no

Limit test:

yes

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day (basis for effect: number of pregnancies) in mice daily treated by feed before, during, and after mating), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (number of pregnancies)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in mice (basis for effect: number of young born, or survival of young animals)), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (number of young born, or survival of young animals).

Key result

Reproductive effects observed:

not specified

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,

- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights, which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

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. Analogua Approach.

CAS Number		Source chemical 77-92-9	Target chemical 631-61-8
CHEMICAL NAME		Citric acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 153 ºC	Experimental data: 114 ºC
Boiling Point		Decomposes	Estimated data: 312.76 ºC
Density		Measured data: 1.665 g/cm3 at 20 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Estimated data: 5.6E-09 mm Hg	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -1.72	Estimated data: -2.79
Water solubility		Measured data: 1330 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 1516 mg/L	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 1535 mg/L	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		Experimental data: (72 h) EC 50 = 640 mg/L	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 = 5790 – 7100 mg/kg bw (mice) LD 50 = 11700 mg/kg bw (rats)	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		Experimental data:   A 4-hour skin irritation study in rabbits exposed to a solution containing 60% citric acid caused erythema and edema. This study did not assess a lethal dose value. TheLD50 was not provided.	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		Experimental data:   Application of 500 mg Citric Acidto rabbit skin produced moderate irritation in 24 hr.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Citric Acidtested on rabbit eyes as single drop of 2% to 5% solution in water caused little or no injury. Irrigation for 30 min with 0.5% to 2% solution causes severe injury; the 0.5% solution causes permanent cloudiness of cornea, and the 2% solution causes severe dense opacification.   Application of 750 mg caused severe irritation in the rabbit eye.	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.
Skin sensitisation		Experimental results:   Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.   No allergic reactions were seen when 60 patients with hand eczema, all of whom were involved in handling food, were patch tested (covered contact, probably 24 hr) with 2.5% citric acid in petrolatum.	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 150-day study with male New Zealand White rabbits daily treated by feed, theNOAEL was 1500 mg/kg bw/day(based on no effects observed at the only dose tested).   In a 6-week study with male Sprague-Dawley rats daily treated by feed, theNOAEL was 4800 mg/kg bw/day(based on no effects observed at the highest 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:   In a bacterial reverse mutation assays usingS. typhimurium(TA97, TA98, TA100 and TA104) in the presence and absence of metabolic activation and up to 2000μg/plate, citric acid was not mutagenic.	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		Experimental data:   In a Dominant Lethal assay using male/female rats dosed at 3 g/kg for 5 days, citric acid did not induced germ cell genotoxicity.	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 data: In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young). In a fertility study, female rats were exposed to citric acid in their daily diet for several months. TheNOAEL (reproductive toxicity) was 600 mg/kg bw/day(based on no effects observed at the only dose tested).   DEVELOPMENTAL TOXICITY / TERATOGENICITY:   Experimental data:   In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young).	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 3009.37 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young animals).

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.01_02 Citric acid) obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in mice (basis for effect: number of pregnancies, number of young born, or survival of young animals)), 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 3009.37 mg/kg bw/day for studied effects.

Reference 5

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles, acceptable for assessment. No GLP.

Principles of method if other than guideline:

Rats were fed diets containing 1.2% citric acid (about 600 mg/kg bw/day). Exposure began 29 weeks prior to mating and continued for a few months after mating. The reproduction of the rats was investigated when they were 32 weeks old and had received the diets for 29 weeks. Eleven weeks later the same animals were mated again.
The average weights for each experimental group at 0, 10 and 20 weeksafter weaning were compared. The effects on the growthg of three succesive generations of rats feed the diet with the substance was investigated. Analysis of blood, teeth, urine, feces, tissues were done.
The blood picture was determined in 20 animals (male/female) of the first generation and in 20 (male/female) of the second generation. Metabolism experimet was performed.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

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 exposure:

Treated rats were fed diets containing 1.2% citric acid.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Several months. Exposure began 29 weeks prior to mating and continued for a few months after mating.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
1.2 % (ca. 600 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

No data

Control animals:

not specified

Positive control:

No data

Parental animals: Observations and examinations:

Fertility of female rats.
The reproduction of the rats was investigated when they were 32 weeks old and had received the diets for 29 weeks. Eleven weeks later the same animals were mated again.

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

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

No reproductive effects were detected.

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (reproductive effects)

Key result

Dose descriptor:

LOAEL

Effect level:

> 600 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (reproductive effects)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Tissues analysis .

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis.

Key result

Dose descriptor:

LOAEL

Effect level:

> 600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Tissue analysis.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis.

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

> 600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Tissue analysis.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis.

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

> 600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis.

Key result

Reproductive effects observed:

not specified

No reproductive effects were detected. The NOAEL for reproductive effects was 600 mg/kg bw/day. The LOAEL was greater than 600 mg/kg bw/day for the same effects.

Conclusions:

The effects on the growthg of three succesive generations of rats feed the diet with the substance were investigated. No reproductive effects were detected in tested generations. The NOAEL for reproductive effects was 600 mg/kg bw/day. The LOAEL was greater than 600 mg/kg bw/day for the same effects. No harmful effects were procuded by the substance in two succesive generations.

Executive summary:

The effects on the growthg of three succesive generations of rats feed the diet with the substance were investigated in a fertility test which was carried out with Citric Acid on rats. Animals were fed diets containing 1.2% citric acid (about 600 mg/kg bw/day). Exposure began 29 weeks prior to mating and continued for a few months after mating. The reproduction of the rats was investigated when they were 32 weeks old and had received the diets for 29 weeks. Eleven weeks later the same animals were mated again.

No reproductive effects were detected. The NOAEL for reproductive effects was 600 mg/kg bw/day. The LOAEL was greater than 600 mg/kg bw/day for the same effects. No harmful effects were procuded by the substance in two succesive generations.

Reference 6

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles, acceptable for assessment. No GLP.

Principles of method if other than guideline:

Rats were fed diets containing 0.1% citric acid, sodium salt. Exposure began 29 weeks prior to mating and continued for a few months after mating. The reproduction of the rats was investigated when they were 32 weeks old and had received the diets for 29 weeks. Eleven weeks later the same animals were mated again.
The average weights for each experimental group at 0, 10 and 20 weeksafter weaning were compared. The effects on the growthg of three succesive generations of rats feed the diet with the substance was investigated. Analysis of blood, teeth, urine, feces, tissues were done.
The blood picture was determined in 20 animals (male/female) of the first generation and in 20 (male/ female) of the second generation. Metabolism experimet was performed.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

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 exposure:

Treated rats were fed diets containing 0.1 citric acid, sodium salt.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Several months. Exposure began 29 weeks prior to mating and continued for a few months after mating.

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:

not specified

Positive control:

No data

Parental animals: Observations and examinations:

Fertility of female rats.
The reproduction of the rats was investigated when they were 32 weeks old and had received the diets for 29 weeks. Eleven weeks later the same animals were mated again.

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

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

No reproductive effects were detected.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (reproductive effects)

Key result

Dose descriptor:

LOAEL

Effect level:

> 50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (reproductive effects)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis: blood, teeth, urine, feces, tissues (kidney, liver, spleen, adrenals, testicles, sceletal muscle and femur).

Key result

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis: blood, teeth, urine, feces, tissues (kidney, liver, spleen, adrenals, testicles, sceletal muscle and femur).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis: blood, teeth, urine, feces, tissues (kidney, liver, spleen, adrenals, testicles, sceletal muscle and femur).

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis: blood, teeth, urine, feces, tissues (kidney, liver, spleen, adrenals, testicles, sceletal muscle and femur).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

no effects obswerved

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis: blood, teeth, urine, feces, tissues (kidney, liver, spleen, adrenals, testicles, sceletal muscle and femur).

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average weights, tissue analysis: blood, teeth, urine, feces, tissues (kidney, liver, spleen, adrenals, testicles, sceletal muscle and femur).

Reproductive effects observed:

not specified

No reproductive effects were detected. The NOAEL for reproductive effects was 50 mg/kg bw/day. The LOAEL was greater than

50 mg/kg bw/day.

Conclusions:

The effects on the growthg of three succesive generations of rats feed the diet with the substance were investigated. No reproductive effects were detected. The NOAEL for reproductive effects was 50 mg/kg bw/day. The LOAEL was greater than 50 mg/kg bw/day.

Executive summary:

The effects on the growthg of three succesive generations of rats feed the diet with Citric Acid sodium salt on rats. Animals were fed diets containing 0.1% citric acid, sodium salt (ca. 50 mg/kg bw/day). Exposure began 29 weeks prior to mating and continued for a few months after mating. The reproduction of the rats was investigated when they were 32 weeks old and had received the diets for 29 weeks. Eleven weeks later the same animals were mated again.

No reproductive effects were detected. The NOAEL for reproductive effects was 50 mg/kg bw/day. The LOAEL was greater than 50 mg/kg bw/day.

Reference 7

Endpoint:

three-generation reproductive toxicity

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 which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 on fertility on the analogue Citric acid.

GLP compliance:

no

Limit test:

no

Clinical signs:

no effects observed

Reproductive performance:

no effects observed

Based on the experimental results obtained with the analogue Citric acid on rats daily treated by feed for several months (NOAEL for reproductive effects = 600 mg/kg bw/day; LOAEL > 600 mg/kg bw/day for the same effects), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be 722.25 mg/kg bw/day, and LOAEL greater than 722.25 mg/kg bw/day for reproductive effects.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

722.25 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Effect level:

> 722.25 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Information from the analogue

Clinical signs:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

722.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Effect level:

> 722.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Clinical signs:

no effects observed

Gross pathological findings:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

722.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

722.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Clinical signs:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

722.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

722.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Reproductive effects observed:

not specified

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,

- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights, which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

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 77-92-9	Target chemical 631-61-8
CHEMICAL NAME		Citric acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 153 ºC	Experimental data: 114 ºC
Boiling Point		Decomposes	Estimated data: 312.76 ºC
Density		Measured data: 1.665 g/cm3 at 20 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Estimated data: 5.6E-09 mm Hg	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -1.72	Estimated data: -2.79
Water solubility		Measured data: 1330 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 1516 mg/L	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 1535 mg/L	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		Experimental data: (72 h) EC 50 = 640 mg/L	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 = 5790 – 7100 mg/kg bw (mice) LD 50 = 11700 mg/kg bw (rats)	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		Experimental data:   A 4-hour skin irritation study in rabbits exposed to a solution containing 60% citric acid caused erythema and edema. This study did not assess a lethal dose value. TheLD50 was not provided.	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		Experimental data:   Application of 500 mg Citric Acidto rabbit skin produced moderate irritation in 24 hr.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Citric Acidtested on rabbit eyes as single drop of 2% to 5% solution in water caused little or no injury. Irrigation for 30 min with 0.5% to 2% solution causes severe injury; the 0.5% solution causes permanent cloudiness of cornea, and the 2% solution causes severe dense opacification.   Application of 750 mg caused severe irritation in the rabbit eye.	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.
Skin sensitisation		Experimental results:   Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.   No allergic reactions were seen when 60 patients with hand eczema, all of whom were involved in handling food, were patch tested (covered contact, probably 24 hr) with 2.5% citric acid in petrolatum.	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 150-day study with male New Zealand White rabbits daily treated by feed, theNOAEL was 1500 mg/kg bw/day(based on no effects observed at the only dose tested).   In a 6-week study with male Sprague-Dawley rats daily treated by feed, theNOAEL was 4800 mg/kg bw/day(based on no effects observed at the highest 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:   In a bacterial reverse mutation assays usingS. typhimurium(TA97, TA98, TA100 and TA104) in the presence and absence of metabolic activation and up to 2000μg/plate, citric acid was not mutagenic.	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		Experimental data:   In a Dominant Lethal assay using male/female rats dosed at 3 g/kg for 5 days, citric acid did not induced germ cell genotoxicity.	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 data: In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young). In a fertility study, female rats were exposed to citric acid in their daily diet for several months. TheNOAEL (reproductive toxicity) was 600 mg/kg bw/day(based on no effects observed at the only dose tested).   DEVELOPMENTAL TOXICITY / TERATOGENICITY:   Experimental data:   In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young).	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:

In the three generations tests the NOAEL with the substance Ammonium acetate is calculated to be 722.25 mg/kg bw/day, and LOAEL greater than 722.25 mg/kg bw/day for reproductive effects in three consecutive generations of rats.

Executive summary:

Based on the experimental results (reported under the endopoint record 07.08.01_05 Citric acid, three generations reproductive study) obtained with the analogue Citric acid on rats daily treated by feed for several months (NOAEL for reproductive effects = 600 mg/kg bw/day; LOAEL > 600 mg/kg bw/day for the same effects), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be 722.25 mg/kg bw/day, and LOAEL greater than 722.25 mg/kg bw/day for reproductive effects.

Reference 8

Endpoint:

three-generation reproductive toxicity

Remarks:

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

Type of information:

migrated 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 toxicity to reproduction 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 Citric acid, sodium salt.

GLP compliance:

no

Limit test:

no

Clinical signs:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Reproductive performance:

no effects observed

Based on the experimental results obtained with the analogue Citric acid, sodium salt, on rats daily treated by feed for several months (NOAEL for reproductive effects = 50 mg/kg bw/day; LOAEL > 50 mg/kg bw/day for the same effects), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be 54 mg/kg bw/day, and LOAEL greater than 54 mg/kg bw/day for reproductive effects.

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 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Effect level:

> 54 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Information from the analogue

Clinical signs:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Effect level:

> 54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

> 54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Clinical signs:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

> 54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Information from the analogue

Reproductive effects observed:

not specified

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.

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 and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye 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.
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 effects on the growthg of three succesive generations of rats feed the diet with the substance were investigated.The NOAEL with the substance Ammonium acetate is calculated to be 54 mg/kg bw/day, and LOAEL greater than 54 mg/kg bw/day for reproductive effects.

Executive summary:

Based on the experimental results (reported under endpoint record 07.08.01_06 Citric acid sodium salt, three generations reproductive study) obtained with the analogue Citric acid, sodium salt, on rats daily treated by feed for several months (NOAEL for reproductive effects = 50 mg/kg bw/day; LOAEL > 50 mg/kg bw/day for the same effects), and the molecular weights, the read-across approach was applied and the NOAEL with the substance Ammonium acetate is calculated to be 54 mg/kg bw/day, and LOAEL greater than 54 mg/kg bw/day for reproductive effects.

Reference 9

Endpoint:

fertility, other

Remarks:

based on test type

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.

Principles of method if other than guideline:

Similar to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).
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

Vehicle:

not specified

Details on exposure:

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

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continuosly

Remarks:

Doses / Concentrations:
0.38 % in diet (222 mg/kg bw/day in males and 239 mg/kg bw/day in females)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0.75 % in diet (441 mg/kg bw/day in males and 484 mg/kg bw/day in females)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1.5 % in diet (886 mg/kg bw/day in males and 961 mg/kg bw/day in females)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
3 % in diet (1792 mg/kg bw/day in males and 1975 mg/kg bw/day in females)
Basis:
nominal in diet

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% .

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), hematocrit (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

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

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.

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.

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, there were no signs indicative of a metabolic acidosis.

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.

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

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

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

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 10

Endpoint:

toxicity to reproduction

Remarks:

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

Type of information:

migrated 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 Ammonium sulfate which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 on fertility on the analogue Ammonium sulfate.

GLP compliance:

not specified

Limit test:

no

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

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.

The analogue Ammonium Sulfate, which shares the same ammonia/ammonium ion functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the aquatic toxicity endpoint. These properties are:
- a low log Pow value which is –5.1 at 25 ºC for Ammonium Sulphate and -2.79 for Ammonium Acetate,
- a similar water solubility which is 764 g/L at 20 ºC for Ammonium Sulphate and 1480 g/L at 4 ºC for Ammonium Acetate, and
- similar molecular weights which are 132.14 for Ammonium Sulphate and 77.08 for Ammonium Acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

1 033.64 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

NOAEL

Effect level:

2 304.12 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

The analogue Ammonium Sulphate, which shares the same ammonia/ammonium ion functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the aquatic toxicity endpoint. These properties are:

- a low log Pow value which is –5.1 at 25 ºC for Ammonium Sulphate and -2.79 for Ammonium Acetate,

- a similar water solubility which is 764 g/L at 20 ºC for Ammonium Sulphate and 1480 g/L at 4 ºC for Ammonium Acetate, and

- similar molecular weights which are 132.14 for Ammonium Sulphate and 77.08 for Ammonium Acetate.

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 7783-20-2	Target chemical 631-61-8
CHEMICAL NAME		Ammonium sulphate	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Experimental data: Decomposes above 280 ºC	Experimental data: 114 ºC
Boiling Point		Experimental data: Decomposes above 280 ºC	Estimated data: 312.76 ºC
Density		Experimental data: 1.769 at 50 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Experimental data: 4.053E-07 Pa (partial pressure of ammonia over solid (NH4)2SO4 at 25 ºC)	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Experimental data: –5.1 at 25 ºC	Estimated data: -2.79
Water solubility		Experimental data: 764 g/L at 20 ºC	Experimental data: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental data: In unsterilized soil, ammonium sulfate is mineralized fairly rapidly, and subsequently nitrified. Nitrification and de-nitrification processes also occur naturally in streams and rivers, as well as in many secondary sewage treatment processes	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		Experimental data: (96 h) LC50 > 100 mg/L (Pimephales promelas)	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		Experimental data: (96 h) LC50 > 100 mg/L (Asellus Intermedius, Daphnia magna, Dugesia tigrina and Gammarus fasciatus)	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		Experimental data: (18 d) EC50 = ca. 25476 mg/L (ca. 2700 mg NH3/L) (Chlorella vulgaris)	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: LC50 >= 2000 mg/kg bw (rat, mouse) LC50 = 3040 mg/kg bw (mouse)	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		Experimental data: (4 h) LC50 > 3.6 mg/m3 (rats) (1 h) LC50 > 2 mg/m3 (rabbits)	No data
Acute Toxicity: Dermal		Experimental data:   LC50 > 2000 mg/kg bw (rats, mice)	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		Experimental data: This substance was not irritating for rabbits. -         Single exposure for 20 hours, intact skin: no skin effects observed. -         Multiple exposures, 8 hrs/day for 5 consecutive days, intact skin: no skin effects observed.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data: This substance was not irritating for rabbits (50 mm3 of undiluted substance).	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.
Skin Sensitization		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		Experimental data: A 14-day inhalation study on rats exposed to 300 mg/m3, the only tested dose, did not report histopathological changes in the lower respiratory tract. As the respiratory tract is the target organ for inhalation exposure, the NOEL for toxicity to the lower respiratory tract is 300 mg/m3.   The NOAEL after feeding diets containing ammonium sulfate for 13 weeks to rats was 886 mg/kg bw/day. The only toxicity sign found was diarrhea in male animals of the high-dose group (LOAEL: 1792 mg/kg bw/day).	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 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: Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems.	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	Experimental data:   Ammonium sulfate did not induce chromosomal aberrations in mammalian or human cell cultures.	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		Experimental data: Similarly to other salts, high doses of ammonium sulfate may have the capability of tumor promotion in the rat stomach; it is, however, much less potent than sodium chloride when tested under identical conditions.	No data
Reproductive Toxicity		TOXICITY TO REPRODUCTION: 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). 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.   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 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 07.08.01_09 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.

No effects on reproductive organs are expected.

Reference 11

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: A scientific study. No GLP.

Principles of method if other than guideline:

From 2 to 4.5 months of age, 80 crossbred gilts were reared in a conventional grower unit where they were naturally exposed to mycoplasmal and bacterial pathogens that cause pneumonia and atrophic rhinitis. At 4.5 months of age, gilts were moved to environmentally regulated rooms (4.9 x 7.3 m) and assigned at random to 1 of 2 treatment groups: low aerial concentration of ammonia (4 to 12 ppm; mean, 7 ppm) or moderate aerial concentration of ammonia (26 to 45 ppm, mean, 35 ppm). Low concentration of ammonia was obtained by flushing of manure pits weekly, whereas moderate concentration of ammonia was maintained by adding anhydrous ammonia to manure pits that were not flushed. Gilts were weighed biweekly. Mean daily gain (MDG) was less (P < 0.01) for gilts exposed to moderate concentration of ammonia than for gilts exposed to low concentration of ammonia after 2 weeks in their respective environments. By 4 and 6 weeks, however, MDG was similar between the 2 treatment groups. After 6 weeks in these environments, 20 gilts from each treatment group were slaughtered, and prevalence and severity of lung lesions and snout grades were determined.

GLP compliance:

no

Limit test:

no

Species:

pig

Strain:

not specified

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4.5 wks

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

female pigs were continuously exposured to 7 and 35 ppm ammonia for 6 weeks before breeding until 30 day of gestation.

Remarks:

Doses / Concentrations:
7 during 6 weeks
Basis:
other: by inhalation

Remarks:

Doses / Concentrations:
35 dirung 6 weeks
Basis:
other: by inhalation

Clinical signs:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Key result

Dose descriptor:

NOAEC

Effect level:

35 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (reproductive effects)

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

No statistically significant differences were noted in ovarian or uterine weights of pigs exposed to about 7 and 35 ppm ammonia for 6 weeks. Female pigs were continuously exposured to 7 and 35 ppm ammonia for 6 weeks before breeding until 30 day of gestation had no statistically significant differences in age at puberty, number of live fetuses, or -to-corpus luteum ration compared to pigs exposed only to 7 ppm. No unexposed controls were included in the study.

Conclusions:

No statistically significant differences were noted in ovarian or uterine weights of pigs exposed to about 7 and 35 ppm ammonia for 6 weeks. Female pigs were continuously exposured to 7 and 35 ppm ammonia for 6 weeks before breeding until 30 day of gestation had no statistically significant differences in age at puberty, number of live fetuses, or -to-corpus luteum ration compared to pigs exposed only to 7 ppm. No unexposed controls were included in the study.

Executive summary:

No statistically significant differences were noted in ovarian or uterine weights of pigs exposed to about 7 and 35 ppm ammonia for 6 weeks. Female pigs were continuously exposured to 7 and 35 ppm ammonia for 6 weeks before breeding until 30 day of gestation had no statistically significant differences in age at puberty, number of live fetuses, or -to-corpus luteum ration compared to pigs exposed only to 7 ppm. No unexposed controls were included in the study.

Reference 12

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: The analogue Ammonium anhydrous which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 on fertility on the analogue Ammonia anhydrous.

GLP compliance:

not specified

Limit test:

no

Clinical signs:

effects observed, treatment-related

Key result

Dose descriptor:

NOEC

Effect level:

157.5 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (reproductive effects)

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

The analogue Ammonia shares the functional group with Ammonium acetate, and also has comparable values for the relevant molecular properties. These properties are:

- a low log Pow value, which is -1.14 at 25 ºC for Ammonia and -2.79 for Ammonium acetate,

- a high water solubility, which is 531 mg/L at 20 ºC for Ammonia and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights, which are 17.03 for Ammonia and 77.08 for Ammonium acetate.

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 7664-41-7	Target chemical 631-61-8
CHEMICAL NAME		Ammonia	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: -77.7 ºC	Experimental data: 114 ºC
Boiling Point		Measured data: -33.35 ºC	Estimated data: 312.76 ºC
Density		Measured data: 0.7710 g/L at 760 mm Hg	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Measured data: 7,510 mm Hg at 25 ºC (from experimentally derived coefficients)	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: - 1.14 at 25 ºC	Estimated data: -2.79
Water solubility		Measured data: 531 mg/L at 20 ºC; 895 mg/L at 0 ºC; 444 mg/L at 28 ºC.	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data:   (96 h) LC50 for Fathead minnows = 8.2 mg/L (hard water, conditions of bioassay not specified) (24-96 h) LC50 for Goldfish = 2 - 2.5 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Coho salmon = 0.45 mg/L (Flow-through bioassay) (72 h) LC50 for Guppy fry = 74 mg/L (Static bioassay) (72 h) LC50 Guppy fry = 1.26 mg/L (Static bioassay) (96 h) LC50 for Cutthroat trout (Salmo clarki) fry = 0.5-0.8 mg/L (Flow-through bioassay) (24 h) LC50 for Rainbow trout fertilized egg > 3.58 mg/L (Static bioassay) (24 h) LC50 for Rainbow trout alevins (0-50 days old) > 3.58 mg/L (Static bioassay) LC50 for Rainbow trout fry (85 days old) = 0.068 mg/L (Static bioassay) (24 h) LC50 for Rainbow trout adult = 0.097 mg/L (Static bioassay) (48 h) LC50 for Walking catfish = 0.28 mg/L (Static bioassay) (96 h) LC50 for Salmo aguabonita = 0.76 mg/L (Conditions of bioassay not specified) (18 h) LC50 for Salmo trutta > 0.15 mg/L (Conditions of bioassay not specified) LC50 for Oncorhynchus tschawytscha = 0.47 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Salvelinus fontinalis = 0.96-1.05 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Proposium williamsoni = 0.47 mg/L (Conditions of bioassay not specified) LC50 for Catostomus platyrhynchos = 0.67-0.82 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Salmo trutta = 0.47 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Oncorhynchus gorbuscha (late alevins) = 0.083 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Oncorhynchus gorbuscha (eyed embryos) > 1.5 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Oncorhynchus kisutch = 0.55 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Salmo salar = 0.28 mg/L (Conditions of bioassay not specified) (96 h) LC50 for Lepomis macrochirus = 0.26-4.6 mg/L (Conditions of bioassay not specified)	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) LC50 for Daphnia magna = 189 mg/L (Static bioassay) (48 h) LC50 for Daphnia magna = 24 mg/L (Conditions of bioassay not specified) (48 h) LC50 for Daphnia pulex = 187 mg/L (Static bioassay) (48 h) LC50 for Ceriodaphnia reticulate = 131 mg/L (Static bioassay) (48 h) LC50 for Simocephalus vetulus = 123 mg/L (Conditions of bioassay not specified)	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: LD50 = 350 mg/kg bw (rat)	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		Experimental data: (1 h) LC50 = 7,050 mg/m3 (rabbit) (1 h) LCLo = 4,900 mg/m3 (rabbit) (1 h) LCLo = 4,900 mg/m3 (cat) (1 h) LC50 = 746 mg/m3 (Cat, dynamic air flow) (Cat) (1 h) LC50 = 7,050 mg/m3 (Cat, static conditions) (2 h) LC50 = 7,600 mg/m3 (rat) (1 h) LC50 = 5,100 mg/m3 (rat) (1 h) LCLo = 1,400 mg/m3 (rat) (10 min) LC50 = 7,105 mg/m3 (mouse) (1 h) LC50 = 3,360 mg/m3 (mouse) (2 h) LC50 = 3,310 mg/m3 (mouse)	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		Experimental data: Ammonia in the form of a gas or an aqueous solution is a recognized skin irritant.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data: Ammonia in the form of a gas or an aqueous solution is a recognized eye irritant.	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.
Skin Sensitization		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: Inhalation   Experimental data:   Rats exposed to 470 mg/m3 continuously found focal or diffuse interstitial pneumonitis in all with epithelial calcification in renal tubules & bronchi, epithelial proliferation of renal tubules, myocardial fibrosis & fatty liver.   49 and 51 rats were exposed continuously for 90 days at 262 mg/m3 and for 65 days at 455 mg/m3 respectively. 262 mg/m3 produced mild nasal discharge in about 25%. All 51 rats exposed at 455mg/m3 showed mild dyspnea & nasal irritation. There were 32 deaths by day 25 and 50by day 65.   One pig exposed to 280 ppm of ammonia showed immediate irritation of the nose and mouth and abnormal respiratory patterns, and by the 36th hour of exposure had convulsions and extremely shallow and irregular breathing. Convulsions continued for 3 hours after exposure ended but the animal appeared normal several hours later. In each of 2 trials, 4 exposure groups of 9 pigs each were continuously exposed to ammonia for 5 weeks. Data from both trials were combined for analysis. Concentrations of ammonia were measured daily, and the average exposures of the groups were 12, 61, 103, and 145 ppm. Feed consumption and average daily weight gain were adversely affected by increasing ammonia concentrations. Pigs exposed to the 3 higher concentrations had excessive nasal, lacrymal, and oral secretions, but these were less pronounced in those exposed to 61 ppm. Pigs exposed to 61 ppm appeared to adjust within 3-4 days, so that their secretory rate was only slightly higher than that of animals exposed to 12 ppm. Pigs in the 2 higher concentrations coughed approximately 3 times as much as those in the lower, and coughing at 61 ppm was slightly more frequent than at 12 ppm. Five animals from each exposure group were autopsied and all gross and microscopic findings were normal.   12 guinea pigs were exposed to about 170 ppm ammonia for 6 hours a day, 5 days a week for up to 18 weeks. Chamber concentrations were monitored and ranged from 140-200 ppm. The exposed animals and 6 controls were weighed weekly. No adverse effects were observed by autopsy of the 4 exposed and 2 control animals killed after 6 weeks or after 12 weeks. In 4 animals exposed for 18 weeks, there was congestion of spleens, livers, and kidneys with early degenerative changes in suprarenal glands. Increased blood destruction was suggested by higher quantities of hemosiderin in the spleens. In the upper tubules of the kidneys there was cloudy swelling with precipitated albumin in the lumen and some casts. These changes were also seen in the lower tubules of 2 animals. The cells of the suprarenal glands were swollen and the cytoplasm in some areas had lost its normal granular structure.   Results of an unstated number of rabbits and cats for 1 hour to initial concentrations of 3.5-8.7 mg/L (approximately 5,200-12,800 ppm) of ammonia with an average concentration of 7.0 mg/L (approximately 10,360 ppm) was reported to be the "approximate LC50." The static method of gassing used probably resulted in an average concentration of half the initial concentrations or less. Also evaluated was the gas absorption of the nasobuccopharyngeal section of the respiratory tract. Rabbits which inhaled directly through a tracheal cannula, and a second group inhaled normally through nose, mouth, and throat. The mean survival time in the second group was reported to be almost twice that of the first group, 33 hr versus 18 hr. On microscopic examination, the trachea was congested and edematous. The mucosa was necrotic and sloughed off in 80-90% of the animals in which the upper respiratory tract had been bypassed, while the trachea was normal in appearance in the second group of test animals. Similar differential findings, but to a lesser degree, were shown in the bronchial mucosa. The damage to the bronchioles and alveoli surprisingly appeared to be identical in both groups. It was described as congestion, edema, hemorrhage, and atelectasis with emphysema. The upper respiratory tract acted as a protection only to the trachea and bronchi, and that small airways and alveoli were less resistant to ammonia injury in many cases within 10 minutes. Between the 6th and 10th postexposure days, 7 of the 80 died, compared with no deaths in controls.Autopsies were not performed.   Pullets exposed to 200 ppm atmospheric ammonia for 17 days had reduced feed intake & reduced growth rate when compared to controls. After the ammonia exposure period at point of lay, percent egg production was less & mortality was greater for exposed group than controls.	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/). Repeated dose toxicity: oral: 15 days study with female Wistar rats. The NOAEL was 3102.2 mg/kg bw/. 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:   Negative in all strains (Salmonella typhimuriumTA98, TA100, TA1535, TA1537, TA1538 andEscherichia coliWP2uvrA) with and without S9 metabolic activation.	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		Experimental data:   Life-long ingestion in drinking water did not produce any carcinogenic effects, and had no effect on the spontaneous development of aderiocarcinoma of the breast in C3H females, a characteristic of this strain.   Male Sprague Dawley rats were exposed for 24 weeks to drinking water containing 83 mg/L N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Subsequently, half of the males were exposed to water containing 0.01% ammonia solution and the other half to tap water for a period of 24 weeks. Chronic oral exposure to a 0.01% ammonia solution via the drinking water exerts a promoting effect on gastric cancer in the rat induced by MNNG. Gross pathology: In the treatment group, 3 out of 37 and in the control group 0 out of 39 rats had metastasis in the liver. The number of rats with gastric tumors was 12 out of 39 in the control group and 26 out of 37 in the treatment group. The number of gastric cancers per tumor bearing rat was significantly higher in ammonia treated rats when compared to control rats, 2.1 and 1.3 respectively. Histopathology: All animals showed signs of gastritis.	No data
Reproductive Toxicity		TOXICITY TO REPRODUCTION: From 2-4.5 months of age, gilts were exposed naturally to Mycoplasma hyponeumoniae and Pasteurella multocida, which cause enzootic pneumonia and atrophic rhinitis, respectively. At 4.5 months of age, the gilts were moved to one of two rooms and exposed to either low (mean 7 ppm) or moderate (mean 36 ppm) aerial concentrations of ammonia. Each exposure group consisted of 40 individuals. In the room with low ammonia concentration, manure was flushed weekly to maintain a 0.3 m depth. In the room with moderate ammonia concentration, manure accumulated to 0.48 m depth. Moderate aerial ammonia concentration was obtained initially and maintained by adding anhydrous ammonia from a steel tank. Mean Daily Gain (MDG) was determined by weighing the gilts biweekly. Half the gilts from each exposure concentration were sacrificed after 6 weeks. The remaining gilts were maintained in their respective environments, exposed daily to mature boars, bred at first estrus, and sacrificed at day 30 of gestation. At the end of two weeks, gilts in the moderate exposure group weighed less than those in the low exposure. After 2 weeks gilts acclimated and the Mean Daily Gain (MDG) was similar for the rest of the experiment. The gilts sacrificed at 6 weeks showed that the animals in the low exposure were heavier. At day 30 of gestation, number of fetuses, fetal length, and fetus-to-corpus luteum ratio were all similar between the two groups.   DEVELPMENTAL 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:

No negative effect are expected at 31.7ppm and 158.5ppm of Ammonium acetate.

Executive summary:

Based on the experimental results (reported under the endopoint record 07.08.01_11 Ammonium) obtained with the analogue,

no statistically significant differences were noted in ovarian or uterine weights of pigs exposed to about 7 and 35 ppm ammonia for 6 weeks. Female pigs were continuously exposured to 7 and 35 ppm ammonia for 6 weeks before breeding until 30 day of gestation had no statistically significant differences in age at puberty, number of live fetuses, or -to-corpus luteum ration compared to pigs exposed only to 7 ppm. No unexposed controls were included in the study.

No negative effect are expected at 31.7ppm and 158.5ppm of Ammonium acetate.

Reference 13

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a two- (or multi-) generation reproductive toxicity study is available

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to column 2 REACH Annex VIII the study is not necessary if or a two-generation reproductive toxicity study is available.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Effect on fertility: via oral route

Dose descriptor:

NOAEL

1 033.64 mg/kg bw/day

Additional information

Weight of evidence: Experimental data on Ammonium Acetate and read-across approach from results obtained with analogues Citric Acid, Sodium Citrate and Ammonium Sulfate on rats and mice.

Read-across from experimental data on Citric Acid:

In the study reported by Wright et al., 1976, a fertility and a one-generation study are described. This study examines the effect of a 5% dietary supplement of Citric acid (ca. 2.5 g/kg bw/day) on the reproductive capacity of rats and mice. Animals were treated before, during, and after mating. No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats and mice with ca. 2.5 g/kg bw/day Citric Acid, compared to controls. The read-across approach is applied and the NOAEL with Ammonium Acetate is calculated to be 3009.37 mg/kg bw/day on rats and mice (basis for effects: number of pregnancies, number of young born, or survival of young).

In the paper reported by Bonting et al., 1956, a fertility test was carried out on rats with Citric Acid. Exposure began 29 weeks prior to mating and continued for a few months after mating. Animals were fed diets containing 1.2 % citric acid (about 600 mg/kg bw/day). No effects were detected. The NOAEL for reproductive effects was 600 mg/kg bw/day. The LOAEL was greater than 600 mg/kg bw/day for the same effects. Applying the read-across approach, the NOAEL with the substance Ammonium acetate is calculated to be 722.25 mg/kg bw/day, and LOAEL greater than 722.25 mg/kg bw/day.

Read-across from experimental data on Citric Acid, sodium salt:

Bonting et al. also describe another fertility test on rats with Citric Acid, sodium salt. The method was exactly the same; the only difference is the given dose. In this case, treated animals received fed diets containing 0.1% citric acid, sodium salt (ca. 50 mg/kg bw/day). No effects were detected. The NOAEL for reproductive effects was 50 mg/kg bw/day. The LOAEL was greater than 50 mg/kg bw/day. Applying the read-across approach, the NOAEL with the substance Ammonium acetate 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 experimental data on Ammonium Sulfate:

In the last paper (Takagi et al., 1999), Fischer 344 rats (10 rats/sex/dose) were exposed during 13 weeks to diets with 0, 222, 441, 886, and 1792 mg/kg bw/day of Ammonium Sulfate in males, and with 0, 239, 484, 961, and 1975 mg/kg bw/day in females. No effects on the reproductive organs were observed. The NOAEL was 886 mg/kg bw/day for males and 1975 mg/kg bw/day for females. 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.

Short description of key information:

Toxicity to reproduction: Weight of evidence: No toxicity to reproduction was observed in any case. Read-across from experimental data on rats and mice treated with Citric Acid, Sodium Citrate, and Ammonium Sulfate.

Effects on developmental toxicity

Description of key information

Developmental toxicity/Teratogenicity: Weight of evidence: No maternal or developmental toxicity was seen in treated animals exposed to high doses of these substances. Experimental data on Ammonium Acetate and read-across from experimental data on rats, mice, and rabbits treated with Sodium Acetate, Citric Acid, Calcium Formate, and Acetic Acid.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No data on GLP. The publication provides scientific valid information as an in vivo teratology screen.

Principles of method if other than guideline:

Pregnant CD-1 mice were generally treated by oral gavage on days 8-12 of gestation at a dose level predicted from a preliminary range finding study to induce a slight degree of maternal toxicity. Day 20 of gestation was considered postnatal day 1 (PD1). On PD1 and 3, the litters were counted and weighed as a unit.

GLP compliance:

not specified

Limit test:

yes

Species:

mouse

Strain:

CD-1

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Time-pregnant female CD-1 mice, approximately 60 days old. The day of sperm plug identification was considered day 1 of pregnancy .

Duration of treatment / exposure:

On Days 8-12 of gestation.

Frequency of treatment:

Daily

Duration of test:

Day 20 of gestation was considered postnatal day 1 (PD1). On PD1 and 3, the litters were counted and weighed as a unit.

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Control: 40 females
Sodium acetate 1000 mg/kg bw/day: 30 females

Control animals:

yes

Maternal examinations:

Mortality, pregnancy and resorption

Fetal examinations:

Mortality and body weight.

Statistics:

Data analysis was performed using the General Linear Models procedure on SAS. When a significant treatment effect was detected by analysis of variance, individual group means were compared using Student's t-test on least-squares means. Since the a priori hypothesis was that treatments could only reduce littex size, a one-tailed test was used for that variable. The number of live pups on PD1 was used as a covariate in the analysis of pup weights.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Details on maternal toxic effects:

Maternal toxic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

mortality

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Remarks:

Neonatal effects at 1st and 3rd days.

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

No maternal toxicity and no neonatal effects were observed with Sodium Acetate at a dose level of 1000 mg/kg bw/day.

Conclusions:

No maternal toxicity and no neonatal effects were observed with Sodium Acetate at a dose level of 1000 mg/kg bw/day.

Executive summary:

Pregnant CD-1 mice were generally treated with Sodium Acetate by oral gavage on days 8-12 of gestation at a dose level predicted from a preliminary range finding study to induce a slight degree of maternal toxicity. Day 20 of gestation was considered postnatal day 1 (PD1). On PD1 and 3, the litters were counted and weighed as a unit.

No maternal toxicity and no neonatal effects (mortality and body weight) were observed at a dose level of 1000 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study meets generally accepted scientific principles, so it is acceptable for assessment. No GLP.

Principles of method if other than guideline:

One-generation study: This study decribes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of the rats. Animals were fed a standard bran/oats dried milk diet. Control groups of female rats were compared to groups of female rats fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet (e.g. ad libitum): Animals were fed a standard bran/oats dried milk diet.

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Animals were treated before, during, and after mating.

Frequency of treatment:

Daily

Duration of test:

No data

Remarks:

Doses / Concentrations:
5% (about 2.5 g/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

6 female animals per group

Control animals:

yes

Maternal examinations:

Number of pregnancies.

Fetal examinations:

Number of young born, or survival of young in rats.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects were seen on number of pregnancies in rats.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

number of abortions

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects were seen on number of young born, or survival of young in rats.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: number of young born, or survival of young

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats with ca. 2.5 g/kg bw/day, compared to controls.

Conclusions:

No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats with ca. 2.5 g/kg bw/day, compared to controls.

Executive summary:

This study decribes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of the rats. Animals were fed a standard bran/oats dried milk diet. Control groups of female rats were compared to groups of female rats fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats with ca. 2.5 g/kg bw/day, compared to controls.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study meets generally accepted scientific principles, so it is acceptable for assessment. No GLP.

Principles of method if other than guideline:

One-generation study: This study decribes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of mice. Animals were fed a standard bran/oats dried milk diet. Control groups of female mice were compared to groups of female mice fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

GLP compliance:

no

Limit test:

yes

Species:

mouse

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet (e.g. ad libitum): Animals were fed a standard bran/oats dried milk diet.

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Animals were treated before, during, and after mating.

Frequency of treatment:

Daily

Duration of test:

No data

Remarks:

Doses / Concentrations:
5% (about 2.5 g/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

6 female animals per group

Control animals:

yes

Maternal examinations:

Number of pregnancies.

Fetal examinations:

Number of young born, or survival of young in mice.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects were seen on number of pregnancies in mice.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

number of abortions

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects were seen on number of young born, or survival of young in mice.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day

Basis for effect level:

other: number of young born, or survival of young

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

No effects were seen on number of pregnancies, number of young born, or survival of young in treated mice with ca. 2.5 g/kg bw/day, compared to controls.

Conclusions:

No effects were seen on number of pregnancies, number of young born, or survival of young in treated mice with ca. 2.5 g/kg bw/day, compared to controls.

Executive summary:

This study decribes an experiment designed to examine the effect of a 5% dietary supplement of citric acid on the reproductive capacity of mice. Animals were fed a standard bran/oats dried milk diet. Control groups of female mice were compared to groups of female mice fed diets containing 5% citric acid (about 2.5 g/kg bw/day) before, during, and after mating. Six animals were in each group for the reproductive studies.

No effects were seen on number of pregnancies, number of young born, or survival of young in treated mice with ca. 2.5 g/kg bw/day, compared to controls.

Reference 4

Endpoint:

developmental toxicity

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 toxicity to reproduction 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 an in vivo teratology screen on the analogue Sodium Acetate.

GLP compliance:

not specified

Limit test:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Data on category approach

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Based on the experimental results obtained with the analogue Sodium Acetate (NOAEL >= 1000 mg/kg bw/day (basis for effect: mortality, pregnancy and resorption) in Pregnant CD-1 mice treated by oral gavage on days 8-12 of gestation), 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 939.66 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 for Sodium Acetate at 25 ºC, 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:

>= 939.66 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 939.66 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data:

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Data on category approach

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Based on the experimental results obtained with the analogue Sodium Acetate (NOAEL >= 1000 mg/kg bw/day (basis for effect: mortality and body weight on neonatals) in Pregnant CD-1 mice treated by oral gavage on days 8-12 of gestation), 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 939.66 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 for Sodium Acetate at 25 ºC, 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:

>= 939.66 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 939.66 mg/kg bw/day (based on maternal toxicity: mortality, pregnancy and resorption; and on neonatal effects: mortality and body weight).

Conclusions:

The NOAEL with the substance Ammonium acetate is calculated to be equal or greater than 939.66 mg/kg bw/day (based on maternal toxicity: mortality, pregnancy and resorption; and on neonatal effects: mortality and body weight).

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.02_01 NaAc) obtained with the analogue Sodium Acetate (NOAEL >= 1000 mg/kg bw/day in Pregnant CD-1 mice treated by oral gavage on days 8-12 of gestation, based on maternal toxicity: mortality, pregnancy and resorption; and on neonatal effects: mortality and body weight), 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 939.66 mg/kg bw/day.

Reference 5

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The analogue Citric Acid which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 one-generation study on the analogue Citric acid.

GLP compliance:

no

Limit test:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Data from analogue

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day (basis for effect: number of pregnancies) in rats daily treated by feed before, during, and after mating), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Data from analogue

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in rats (basis for effect: number of young born, or survival of young animals)), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Basis for effect level:

other: RA from analogue ( number of young born, or survival of young)

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,

- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights, which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

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 77-92-9	Target chemical 631-61-8
CHEMICAL NAME		Citric acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 153 ºC	Experimental data: 114 ºC
Boiling Point		Decomposes	Estimated data: 312.76 ºC
Density		Measured data: 1.665 g/cm3 at 20 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Estimated data: 5.6E-09 mm Hg	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -1.72	Estimated data: -2.79
Water solubility		Measured data: 1330 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 1516 mg/L	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 1535 mg/L	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		Experimental data: (72 h) EC 50 = 640 mg/L	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 = 5790 – 7100 mg/kg bw (mice) LD 50 = 11700 mg/kg bw (rats)	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		Experimental data:   A 4-hour skin irritation study in rabbits exposed to a solution containing 60% citric acid caused erythema and edema. This study did not assess a lethal dose value. TheLD50 was not provided.	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		Experimental data:   Application of 500 mg Citric Acidto rabbit skin produced moderate irritation in 24 hr.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Citric Acidtested on rabbit eyes as single drop of 2% to 5% solution in water caused little or no injury. Irrigation for 30 min with 0.5% to 2% solution causes severe injury; the 0.5% solution causes permanent cloudiness of cornea, and the 2% solution causes severe dense opacification.   Application of 750 mg caused severe irritation in the rabbit eye.	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		Experimental results:   Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.   No allergic reactions were seen when 60 patients with hand eczema, all of whom were involved in handling food, were patch tested (covered contact, probably 24 hr) with 2.5% citric acid in petrolatum.	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 150-day study with male New Zealand White rabbits daily treated by feed, theNOAEL was 1500 mg/kg bw/day(based on no effects observed at the only dose tested).   In a 6-week study with male Sprague-Dawley rats daily treated by feed, theNOAEL was 4800 mg/kg bw/day(based on no effects observed at the highest 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:   In a bacterial reverse mutation assays usingS. typhimurium(TA97, TA98, TA100 and TA104) in the presence and absence of metabolic activation and up to 2000μg/plate, citric acid was not mutagenic.	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		Experimental data:   In a Dominant Lethal assay using male/female rats dosed at 3 g/kg for 5 days, citric acid did not induced germ cell genotoxicity.	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 data: In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young). In a fertility study, female rats were exposed to citric acid in their daily diet for several months. TheNOAEL (reproductive toxicity) was 600 mg/kg bw/day(based on no effects observed at the only dose tested).   DEVELOPMENTAL TOXICITY / TERATOGENICITY:   Experimental data:   In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young).	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 3009.37 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young animals).

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.02_02 Citric acid) obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in rats, basis for effect: number of pregnancies, number of young born, or survival of young animals), 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 3009.37 mg/kg bw/day for studied effects.

Reference 6

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The analogue Citric Acid which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 one-generation study on the analogue Citric acid.

GLP compliance:

no

Limit test:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Data on analogue

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day (basis for effect: number of pregnancies) in mice daily treated by feed before, during, and after mating), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Data on analogue

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Based on the experimental results obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in mice (basis for effect: number of young born, or survival of young animals)), 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 3009.37 mg/kg bw/day.

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,
- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 009.37 mg/kg bw/day

Basis for effect level:

other: RA from analogue (number of young born, or survival of young).

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The analogue Citric acid 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 -1.72 for Citric acid, and -2.79 for Ammonium acetate,

- a high water solubility, which is 1330 g/L for Citric acid, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights, which are 192.1 for Citric acid, and 77.08 for Ammonium acetate.

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 77-92-9	Target chemical 631-61-8
CHEMICAL NAME		Citric acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 153 ºC	Experimental data: 114 ºC
Boiling Point		Decomposes	Estimated data: 312.76 ºC
Density		Measured data: 1.665 g/cm3 at 20 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Estimated data: 5.6E-09 mm Hg	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -1.72	Estimated data: -2.79
Water solubility		Measured data: 1330 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 1516 mg/L	Experimental data and read-across from Potassium Acetate, based on molecular weights:   LC50 = 392.70 mg/L.
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 1535 mg/L	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		Experimental data: (72 h) EC 50 = 640 mg/L	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 = 5790 – 7100 mg/kg bw (mice) LD 50 = 11700 mg/kg bw (rats)	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		Experimental data:   A 4-hour skin irritation study in rabbits exposed to a solution containing 60% citric acid caused erythema and edema. This study did not assess a lethal dose value. TheLD50 was not provided.	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		Experimental data:   Application of 500 mg Citric Acidto rabbit skin produced moderate irritation in 24 hr.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate, Ammonium Sulphate, and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Citric Acidtested on rabbit eyes as single drop of 2% to 5% solution in water caused little or no injury. Irrigation for 30 min with 0.5% to 2% solution causes severe injury; the 0.5% solution causes permanent cloudiness of cornea, and the 2% solution causes severe dense opacification.   Application of 750 mg caused severe irritation in the rabbit eye.	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		Experimental results:   Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.   No allergic reactions were seen when 60 patients with hand eczema, all of whom were involved in handling food, were patch tested (covered contact, probably 24 hr) with 2.5% citric acid in petrolatum.	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 150-day study with male New Zealand White rabbits daily treated by feed, theNOAEL was 1500 mg/kg bw/day(based on no effects observed at the only dose tested).   In a 6-week study with male Sprague-Dawley rats daily treated by feed, theNOAEL was 4800 mg/kg bw/day(based on no effects observed at the highest 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:   In a bacterial reverse mutation assays usingS. typhimurium(TA97, TA98, TA100 and TA104) in the presence and absence of metabolic activation and up to 2000μg/plate, citric acid was not mutagenic.	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		Experimental data:   In a Dominant Lethal assay using male/female rats dosed at 3 g/kg for 5 days, citric acid did not induced germ cell genotoxicity.	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 data: In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young). In a fertility study, female rats were exposed to citric acid in their daily diet for several months. TheNOAEL (reproductive toxicity) was 600 mg/kg bw/day(based on no effects observed at the only dose tested).   DEVELOPMENTAL TOXICITY / TERATOGENICITY:   Experimental data:   In a one-generation oral reproductive toxicity study, female rats and mice were daily treated with citric acid before, during, and after mating. The NOAEL was equal or greater than 2500 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young).	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 3009.37 mg/kg bw/day (basis for effect: number of pregnancies, number of young born, or survival of young animals).

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.02_03 Citric acid) obtained with the analogue Citric acid (NOAEL >= 2500 mg/kg bw/day in mice; basis for effect: number of pregnancies, number of young born, or survival of young animals), 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 3009.37 mg/kg bw/day for studied effects.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. No GLP.

Principles of method if other than guideline:

A three-generation drinking water study at 0 or 200 mg/kg bw/day Calcium Formate in the drinking water was performed in Wistar rats. The duration of the test was ca. 3 years. At the beginning of the test, 8 males and 24 females in the treated group, and 8 males and 8 females in the control group were used. Number, weight and length of offspring were examined. A portion of the offspring was also sacrificed shortly after birth for evaluation of developmental toxicity.

GLP compliance:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 160-200 g

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

No data

Duration of treatment / exposure:

A three-generation study.

Frequency of treatment:

Daily

Duration of test:

ca. 3 years

Remarks:

Doses / Concentrations:
200 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

At the beginning of the test:
8 males and 24 females in the treated group
8 males and 8 females in the control group

Control animals:

yes

Fetal examinations:

Number, weight and length of offspring were examined.
A portion of the offspring was also sacrificed shortly after birth for evaluation of developmental toxicity.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects on fertility (up to 5 generations) were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number, weight and length of offspring did not differ in treated animals from controls.
No statistical differences in organ or bone abnormalities were found.
The growth of treated offspring was similar to controls.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: growth, weight and lenght

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The NOAEL for maternal and developmental toxicity was equal or higher than 200 mg/kg bw/day.

Conclusions:

The NOAEL for maternal and developmental toxicity was equal or higher than 200 mg/kg bw/day.

Executive summary:

A three-generation drinking water study at 0 or 200 mg/kg bw/day Calcium Formate in the drinking water was performed in Wistar rats. The duration of the test was ca. 3 years. At the beginning of the test, 8 males and 24 females in the treated group, and 8 males and 8 females in the control group were used.

No effects on fertility were observed. Number, weight and length of offspring did not differ in treated animals from controls.A portion of the offspring was also sacrificed shortly after birth for evaluation of developmental toxicity. No statistical differences in organ or bone abnormalities were found. The growth of treated offspring was similar to controls.

The NOAEL for maternal and developmental toxicity was equal or higher than 200 mg/kg bw/day.

Reference 8

Endpoint:

developmental toxicity

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 Calcium formate which has a similar functional group to Ammonium Acetate (CH3COO- vs. HCOO-), also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 three-generation study on the analogue Calcium formate.

GLP compliance:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Data from analogue

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Based on the experimental results obtained with the analogue Calcium formate (NOAEL >= 200 mg/kg bw/day in Wistar rats for maternal toxicity, fertility), 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 236.96 mg/kg bw/day.

The analogue Calcium formate which has a similar functional group to Ammonium Acetate (CH3COO- vs. HCOO-), also has comparable values for the relevant molecular properties. These properties are:
- a low log Pow value which is -2.47 for Calcium formate, and -2.79 for Ammonium acetate,
- a high water solubility, which is 166 g/L for Calcium formate at 20 ºC, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights, which are 130.113 for Calcium formate, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 236.96 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 236.96 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Data from analogue

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Based on the experimental results obtained with the analogue Calcium formate (NOAEL >= 200 mg/kg bw/day in Wistar rats for developmental toxicity, overall effects), 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 236.96 mg/kg bw/day.

The analogue Calcium formate which has a similar functional group to Ammonium Acetate (CH3COO- vs. HCOO-), also has comparable values for the relevant molecular properties. These properties are:
- a low log Pow value which is -2.47 for Calcium formate, and -2.79 for Ammonium acetate,
- a high water solubility, which is 166 g/L for Calcium formate at 20 ºC, and 1480 g/L at 4 ºC for Ammonium acetate, and
- similar molecular weights, which are 130.113 for Calcium formate, and 77.08 for Ammonium acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 236.96 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: growth, weight and lenght

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The analogue Calcium formate which has a similar functional group to Ammonium Acetate (CH3COO- vs. HCOO-), also has comparable values for the relevant molecular properties. These properties are:

 - a low log Pow value which is -2.47 for Calcium formate, and -2.79 for Ammonium acetate,

 - a high water solubility, which is 166 g/L for Calcium formate at 20 ºC, and 1480 g/L at 4 ºC for Ammonium acetate, and

 - similar molecular weights, which are 130.113 for Calcium formate, and 77.08 for Ammonium acetate.

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. Analogua Approach.

CAS Number		Source chemical 544-17-2	Target chemical 631-61-8
CHEMICAL NAME		Calcium formate	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: > 300 ºC	Experimental data: 114 ºC
Boiling Point		Measured data: > 300 ºC	Estimated data: 312.76 ºC
Density		Measured data: Relative density: 2.015	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: -2.47	Estimated data: -2.79
Water solubility		Measured data: 160 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Readily biodegradable	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		Experimental data: (96 h) LC 50 > 1000 mg/L	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 = 2650 mg/kg bw (rats) LD 50 = 1920 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 and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye 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.
Skin Sensitization		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		A lifetime drinking water study has been conducted with calcium formate in the drinking water at 0, 0.2, or 0.4% (150-200 mg/kg/day in the lowest dose according to the authors). Six rats per group were. Macroscopic and histological examinations were conducted upon the natural death of the animals. No significant clinical or pathologic changes (growth or organ functions) were detected in any dose group; in particular, there were no disorders of the ocular fundus. The study includes several generations (up to 5). At the beginning, 8 males and 24 females were used.	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/. 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:   Calcium formate hasbeen found negative in the Ames test, with and without metabolic activation.	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:   No data     DEVELOPMENTAL TOXICITY / TERATOGENICITY:   Experimental data:   Results of a three-generation drinking water study at 0 or 200 mg/kg/day calcium formate in the drinking water have been published. Number, weight and length of offspring did not differ in treated animals from controls. In this study, a portion of the offspring was also sacrificed shortly after birth for evaluation of developmental toxicity. No statistical differences in organ or bone abnormalities were found. The growth of treated offspring was similar to controls.	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 236.96 mg/kg bw/day for maternal and developmental toxicity (overall effects).

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.02_07 Calcium formate) obtained with the analogue Calcium formate (NOAEL >= 200 mg/kg bw/day in Wistar rats for maternal and developmental toxicity), 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 236.96 mg/kg bw/day.

Reference 9

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A FDA study.

Principles of method if other than guideline:

Following mating, adult female albino CD-1 mice were dosed daily by oral intubation beginning on day 6 of gestation. Animals were observed daily and body weights recorded for 10 days. On day 17, Caesarian sections were performed on all dams and the numbers of implantation sites, resorption sites, and live and dead fetuses was recorded. General external and internal examinations were also made of the dams.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 10 days (beginning on day 6 of gestation).

Frequency of treatment:

Daily

Duration of test:

17 days

Remarks:

Doses / Concentrations:
16 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
74 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
345 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1600 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

Only female animals were used.

Control animals:

yes, concurrent no treatment

Maternal examinations:

Animals were observed daily and body weights recorded for 10 days.

Ovaries and uterine content:

On day 17, Caesarian sections were performed on all dams and the numbers of implantation sites and resorption sites was recorded.

Fetal examinations:

After cesarea, the numbers of live and dead fetuses was recorded.
General external and internal examinations were also made of the dams.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects on nidation or on maternal survival were observed at doses up to 1600 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects on fetal survival were observed at doses up to 1600 mg/kg bw/day.
The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

No effects on nidation or on maternal or fetal survival were observed at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Conclusions:

No effects on nidation or on maternal or fetal survival were observed at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Executive summary:

Following mating, adult female albino CD-1 mice were dosed daily by oral intubation beginning on day 6 of gestation. Tested doses were 0, 16, 74, 345, and 1600 mg/kg bw/day. Animals were observed daily and body weights recorded for 10 days. On day 17, Caesarian sections were performed on all dams and the numbers of implantation sites, resorption sites, and live and dead fetuses was recorded. General external and internal examinations were also made of the dams.

No effects on nidation or on maternal or fetal survival were observed at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Reference 10

Endpoint:

developmental toxicity

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 Acetic Acid which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 one-generation study on the analogue Acetic Acid.

GLP compliance:

no

Limit test:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Data from analogue

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Based on the experimental results obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female CD-1 mice treated for 10 days for maternal toxicity, mortality and body weight gain), 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 2055.47 mg/kg bw/day.

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,
- a high water solubility which is 50 g/L for Acetic acid and 1480 g/L for Ammonium Acetate at 4 ºC, and
- similar molecular weights which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 055.47 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 055.47 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Data from analogue

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Based on the experimental results obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female CD-1 mice treated for 10 days for developmental toxicity, numbers of live and dead fetuses, external and internal examinations), 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 2055.47 mg/kg bw/day.

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,
- a high water solubility which is 50 g/L for Acetic acid and 1480 g/L for Ammonium Acetate at 4 ºC, and
- similar molecular weights which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 480 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,

- a high water solubility, which is 50 g/L for Acetic acid and 1480 g/L at 4 ºC for Ammonium Acetate, and

- similar molecular weights, which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

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 64-19-7	Target chemical 631-61-8
CHEMICAL NAME		Acetic acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 16.7 ºC	Experimental data: 114 ºC
Boiling Point		Measured data: 118.1 ºC	Estimated data: 312.76 ºC
Density		Measured data: 1.0446 g/cm3 at 25 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Measured data: 11.4 mm Hg at 20 °C	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -0.17	Estimated data: -2.79
Water solubility		Measured data: 50 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 251 mg/L(Gambusia affinis)	Experimental data and read-across from Potassium Acetate, based on molecular weights: LC50=392.70mg/L
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 65 mg/L(Daphnia magna)	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		Experimental data:   (8 d) Toxicity threshold (TT) = 4000 mg/L (Scenedesmus quadricauda)	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 = 3250-8610 mg/kg bw (rats) LD 50 = 4960 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		Experimental results: (4 h) LC50 = 11.4 mg/L	No data
Acute Toxicity: Dermal		Experimental results: LD 50 = 1060 mg/kg bw (rabbits)	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		Experimental data:   No effect was found in guinea pigs or rabbits after application of 10% acetic acid solution to intact or abraded skin patches. Concentrations from 80% to glacial produced severe burns to guinea pig skin, concentrations form 50-80% produced moderate to severe burns, and below 50% there was relatively mild injury.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate, Ammonium Sulphate, and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Liquid glacial acetic acid causes devastating injury when applied to the eyes of rabbits. Acetic acid penetrates intact corneal epithelium rapidly and reaches iris in concentrations high enough to cause iritis.	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 Sensitization		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:   8-month study with male rats treated by gavage 3 times per week. TheLOAEL = 60 mg/kg bw/day(based on hyperplasia of the esophagus and forestomach, indicative of irritation at point of contact, only one dose tested). The NOAEL = Not established(only one dose tested).   Repeated dose toxicity: inhalation:   35-day study with rats and mice. The NOAEL was ca. 0.09 mg/L/day(based on no evidence of adverse effects at any dose tested).	Repeated dose toxicity: oral: Weight of evidence: Experimental results:   Repeated dose toxicity: oral: 90 days withfemale Wistar rats. The NOAEL 3150.4 mg/kg bw/day. Repeated dose toxicity: oral: 15 days study with female Wistar rats. The 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 results:   In a bacterial reverse mutation assay using Salmonella typhimurium(TA98, TA100, TA1535, TA97 and TA1537) in the presence and absence of metabolic activation and up to 10000μg/plate, acetic acid was not mutagenic.	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 Phenoxyacetic 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	Experimental results:   Acetic acid was not clastogenic in anin vitroassay using Chinese hamster ovary K1 cells at concentrations < 16 mM; 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:   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: No data   DEVELPMENTAL TOXICITY / TERATOGENICITY: A series of developmental toxicity studies were conducted in CD-1 mice, Wistar rats, and Dutch-belted rabbits. Pregnant females were daily administered acetic acid by gavage on gestation day 6 – 16 for rats and mice, and 6 – 19 for rabbits. The NOAEL (maternal and developmental toxicity) (rats, mice, and rabbits) = 1600 mg/kg bw/day (based on no effects observed at the highest dose tested).	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 postnatal 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/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 2055.47 mg/kg bw/day for maternal and developmental toxicity in mice.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.02_09 Acetic acid) obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female CD-1 mice treated for 10 days for maternal toxicity, mortality and body weight gain, and for developmental toxicity, numbers of live and dead fetuses, external and internal examinations), 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 2055.47 mg/kg bw/day for maternal and developmental toxicity.

Reference 11

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A FDA study.

Principles of method if other than guideline:

Following mating, adult female albino rats (Wistar) were dosed daily by oral intubation beginning on day 6 of gestation. Animals were observed daily and body weights recorded. On day 20, Caesarian sections were performed on all dams and the numbers of implantation sites, resorption sites, and live and dead fetuses was recorded. General external and internal examinations were also made of the dams.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 10 days (beginning on day 6 of gestation).

Frequency of treatment:

Daily

Duration of test:

14 days

Remarks:

Doses / Concentrations:
16 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
74 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
345 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1600 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

Only female animals were used.

Control animals:

yes, concurrent no treatment

Maternal examinations:

Animals were observed daily and body weights recorded.

Ovaries and uterine content:

On day 20, Caesarian sections were performed on all dams and the numbers of implantation sites and resorption sites was recorded.

Fetal examinations:

After cesarea, the numbers of live and dead fetuses was recorded.
General external and internal examinations were also made of the dams.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects on nidation or on maternal survival were observed at doses up to 1600 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects on fetal survival were observed at doses up to 1600 mg/kg bw/day.
The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

No effects on nidation or on maternal or fetal survival at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Conclusions:

No effects on nidation or on maternal or fetal survival at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Executive summary:

Following mating, adult female albino rats (Wistar) were dosed daily by oral intubation beginning on day 6 of gestation. Tested doses were 0, 16, 74, 345, and 1600 mg/kg bw/day. Animals were observed daily and body weights recorded. On day 20, Caesarian sections were performed on all dams and the numbers of implantation sites, resorption sites, and live and dead fetuses was recorded. General external and internal examinations were also made of the dams.

No effects on nidation or on maternal or fetal survival at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Reference 12

Endpoint:

developmental toxicity

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 Acetic Acid which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 one-generation study on the analogue Acetic Acid.

GLP compliance:

no

Limit test:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Data from analogue

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Based on the experimental results obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female Wistar rats treated for 10 days for maternal toxicity, mortality and body weight gain), 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 2055.47 mg/kg bw/day.

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,
- a high water solubility which is 50 g/L for Acetic acid and 1480 g/L for Ammonium Acetate at 4 ºC, and
- similar molecular weights which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 055.47 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 055.47 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: RA data: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Data from analogue

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Based on the experimental results obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female Wistar rats treated for 10 days for developmental toxicity, numbers of live and dead fetuses, external and internal examinations), 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 2055.47 mg/kg bw/day.

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,
- a high water solubility which is 50 g/L for Acetic acid and 1480 g/L for Ammonium Acetate at 4 ºC, and
- similar molecular weights which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 055.47 mg/kg bw/day

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,

- a high water solubility, which is 50 g/L for Acetic acid and 1480 g/L at 4 ºC for Ammonium Acetate, and

- similar molecular weights, which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

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 64-19-7	Target chemical 631-61-8
CHEMICAL NAME		Acetic acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 16.7 ºC	Experimental data: 114 ºC
Boiling Point		Measured data: 118.1 ºC	Estimated data: 312.76 ºC
Density		Measured data: 1.0446 g/cm3 at 25 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Measured data: 11.4 mm Hg at 20 °C	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -0.17	Estimated data: -2.79
Water solubility		Measured data: 50 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 251 mg/L(Gambusia affinis)	Experimental data and read-across from Potassium Acetate, based on molecular weights: LC50=392.70mg/L
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 65 mg/L(Daphnia magna)	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		Experimental data:   (8 d) Toxicity threshold (TT) = 4000 mg/L (Scenedesmus quadricauda)	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 = 3250-8610 mg/kg bw (rats) LD 50 = 4960 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		Experimental results: (4 h) LC50 = 11.4 mg/L	No data
Acute Toxicity: Dermal		Experimental results: LD 50 = 1060 mg/kg bw (rabbits)	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		Experimental data:   No effect was found in guinea pigs or rabbits after application of 10% acetic acid solution to intact or abraded skin patches. Concentrations from 80% to glacial produced severe burns to guinea pig skin, concentrations form 50-80% produced moderate to severe burns, and below 50% there was relatively mild injury.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Liquid glacial acetic acid causes devastating injury when applied to the eyes of rabbits. Acetic acid penetrates intact corneal epithelium rapidly and reaches iris in concentrations high enough to cause iritis.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate , Ammonium Sulpha and Ammonium Stearate, based on functional group: The substance Ammonium Acetate is considered as not irritating for eyes.
Skin Sensitization		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:   8-month study with male rats treated by gavage 3 times per week. TheLOAEL = 60 mg/kg bw/day(based on hyperplasia of the esophagus and forestomach, indicative of irritation at point of contact, only one dose tested). The NOAEL = Not established(only one dose tested).   Repeated dose toxicity: inhalation:   35-day study with rats and mice. The NOAEL was ca. 0.09 mg/L/day(based on no evidence of adverse effects at any dose tested).	Repeated dose toxicity: oral: Weight of evidence: Experimental results:   Repeated dose toxicity: oral: 90 days withfemale Wistar rats. The NOAEL 3150.4 mg/kg bw/day. Repeated dose toxicity: oral: 15 days study with female Wistar rats. The 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 results:   In a bacterial reverse mutation assay using Salmonella typhimurium(TA98, TA100, TA1535, TA97 and TA1537) in the presence and absence of metabolic activation and up to 10000μg/plate, acetic acid was not mutagenic.	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 Phenoxyacetic 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	Experimental results:   Acetic acid was not clastogenic in anin vitroassay using Chinese hamster ovary K1 cells at concentrations < 16 mM; 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:   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: No data   DEVELPMENTAL TOXICITY / TERATOGENICITY: A series of developmental toxicity studies were conducted in CD-1 mice, Wistar rats, and Dutch-belted rabbits. Pregnant females were daily administered acetic acid by gavage on gestation day 6 – 16 for rats and mice, and 6 – 19 for rabbits. The NOAEL (maternal and developmental toxicity) (rats, mice, and rabbits) = 1600 mg/kg bw/day (based on no effects observed at the highest dose tested).	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 postnatal 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/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 2055.47 mg/kg bw/day for maternal and developmental toxicity in rats.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.02_11 Acetic acid) obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female Wistar rats treated for 10 days for maternal toxicity, mortality and body weight gain, and for developmental toxicity, numbers of live and dead fetuses, external and internal examinations), 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 2055.47 mg/kg bw/day for maternal and developmental toxicity.

Reference 13

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A FDA study.

Principles of method if other than guideline:

Following artificial insemination, adult Dutch-belted female rabbits were dosed daily by oral intubation beginning on day 6 of gestation. Animals were observed daily and body weights recorded. On day 29, Caesarian sections were performed on all does and the numbers of corpora lutea, implantation sites, resorption sites, and live and dead fetuses was recorded. General external and internal examinations were also made of the does.

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

Dutch

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Female rabbits were artificially inseminated.

Duration of treatment / exposure:

Exposure period: 13 days (beginning on day 6 of gestation).

Frequency of treatment:

Daily

Duration of test:

23 days

Remarks:

Doses / Concentrations:
16 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
74 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
345 mg/kg bw/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1600 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

Only female animals were used.

Control animals:

yes, concurrent no treatment

Maternal examinations:

Animals were observed daily and body weights recorded.

Ovaries and uterine content:

On day 29, Caesarian sections were performed on all does and the numbers of corpora lutea, implantation sites and resorption sites was recorded.

Fetal examinations:

After cesarea, the numbers of live and dead fetuses was recorded.
General external and internal examinations were also made of the does.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects on nidation or on maternal survival were observed at doses up to 1600 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects on fetal survival were observed at doses up to 1600 mg/kg bw/day.
The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

No effects on nidation or on maternal or fetal survival at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Conclusions:

No effects on nidation or on maternal or fetal survival at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Executive summary:

Following artificial insemination, adult Dutch-belted female rabbits were dosed daily by oral intubation beginning on day 6 of gestation. Tested doses were 0, 16, 74, 345, and 1600 mg/kg bw/day. Animals were observed daily and body weights recorded. On day 29, Caesarian sections were performed on all does and the numbers of corpora lutea, implantation sites, resorption sites, and live and dead fetuses was recorded. General external and internal examinations were also made of the does.

No effects on nidation or on maternal or fetal survival at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls.

Reference 14

Endpoint:

developmental toxicity

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 Acetic Acid which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the toxicity to reproduction 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 one-generation study on the analogue Acetic Acid.

GLP compliance:

no

Limit test:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Data from analogue

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Based on the experimental results obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female Dutch-belted rabbits treated for 13 days for maternal toxicity, mortality and body weight gain), 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 2055.47 mg/kg bw/day.

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,
- a high water solubility which is 50 g/L for Acetic acid and 1480 g/L for Ammonium Acetate at 4 ºC, and
- similar molecular weights which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 055.47 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 055.47 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Based on the experimental results obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female Dutch-belted rabbits treated for 13 days for developmental toxicity, numbers of live and dead fetuses, external and internal examinations), 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 2055.47 mg/kg bw/day.

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,
- a high water solubility which is 50 g/L for Acetic acid and 1480 g/L for Ammonium Acetate at 4 ºC, and
- similar molecular weights which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

Key result

Dose descriptor:

NOAEL

Effect level:

2 055.47 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The analogue Acetic acid 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 -0.17 for Acetic acid and -2.79 for Ammonium Acetate,

- a high water solubility, which is 50 g/L for Acetic acid and 1480 g/L at 4 ºC for Ammonium Acetate, and

- similar molecular weights, which are 60.0 for Acetic acid and 77.08 for Ammonium Acetate.

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 64-19-7	Target chemical 631-61-8
CHEMICAL NAME		Acetic acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Measured data: 16.7 ºC	Experimental data: 114 ºC
Boiling Point		Measured data: 118.1 ºC	Estimated data: 312.76 ºC
Density		Measured data: 1.0446 g/cm3 at 25 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Measured data: 11.4 mm Hg at 20 °C	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Measured data: -0.17	Estimated data: -2.79
Water solubility		Measured data: 50 g/L	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental results: Readily biodegradable	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		Experimental data: (96 h) LC 50 = 251 mg/L(Gambusia affinis)	Experimental data and read-across from Potassium Acetate, based on molecular weights: LC50=392.70mg/L
Acute Toxicity to Aquatic Invertebrates		Experimental data: (48 h) EC 50 = 65 mg/L(Daphnia magna)	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		Experimental data:   (8 d) Toxicity threshold (TT) = 4000 mg/L (Scenedesmus quadricauda)	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 = 3250-8610 mg/kg bw (rats) LD 50 = 4960 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		Experimental results: (4 h) LC50 = 11.4 mg/L	No data
Acute Toxicity: Dermal		Experimental results: LD 50 = 1060 mg/kg bw (rabbits)	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		Experimental data:   No effect was found in guinea pigs or rabbits after application of 10% acetic acid solution to intact or abraded skin patches. Concentrations from 80% to glacial produced severe burns to guinea pig skin, concentrations form 50-80% produced moderate to severe burns, and below 50% there was relatively mild injury.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate and Ammonium Lactate, and Ammonium Stearate based on functional group: The substance Ammonium Acetate is considered as not irritating for skin.
Eye Irritation/Corrosion		Experimental data:   Liquid glacial acetic acid causes devastating injury when applied to the eyes of rabbits. Acetic acid penetrates intact corneal epithelium rapidly and reaches iris in concentrations high enough to cause iritis.	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.
Skin Sensitization		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:   8-month study with male rats treated by gavage 3 times per week. TheLOAEL = 60 mg/kg bw/day(based on hyperplasia of the esophagus and forestomach, indicative of irritation at point of contact, only one dose tested). The NOAEL = Not established(only one dose tested).   Repeated dose toxicity: inhalation:   35-day study with rats and mice. The NOAEL was ca. 0.09 mg/L/day(based on no evidence of adverse effects at any dose tested).	Repeated dose toxicity: oral: Weight of evidence: Experimental results:   Repeated dose toxicity: oral: 90 days withfemale Wistar rats. The NOAEL 3150.4 mg/kg bw/day. Repeated dose toxicity: oral: 15 days study with female Wistar rats. The 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 results:   In a bacterial reverse mutation assay using Salmonella typhimurium(TA98, TA100, TA1535, TA97 and TA1537) in the presence and absence of metabolic activation and up to 10000μg/plate, acetic acid was not mutagenic.	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 Phenoxyacetic 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	Experimental results:   Acetic acid was not clastogenic in anin vitroassay using Chinese hamster ovary K1 cells at concentrations < 16 mM; 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:   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: No data   DEVELPMENTAL TOXICITY / TERATOGENICITY: A series of developmental toxicity studies were conducted in CD-1 mice, Wistar rats, and Dutch-belted rabbits. Pregnant females were daily administered acetic acid by gavage on gestation day 6 – 16 for rats and mice, and 6 – 19 for rabbits. The NOAEL (maternal and developmental toxicity) (rats, mice, and rabbits) = 1600 mg/kg bw/day (based on no effects observed at the highest dose tested).	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 postnatal 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/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 2055.47 mg/kg bw/day for maternal and developmental toxicity in rabbits.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.08.02_13 Acetic acid) obtained with the analogue Acetic acid (NOAEL >= 1600 mg/kg bw/day in female Dutch-belted rabbits treated for 13 days for maternal toxicity, mortality and body weight gain, and for developmental toxicity, numbers of live and dead fetuses, external and internal examinations), 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 2055.47 mg/kg bw/day for maternal and developmental toxicity.

Reference 15

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Test method was not according to any guideline. No data on GLP.

Principles of method if other than guideline:

Wistar rats were used to study the toxicity effects of prenatal exposure of Ammonium Acetate. For prenatal exposure to ammonia, female rats were made hyperammonemic by feeding them a diet containing Ammonium acetate (4293 mg ammonium/kg bw/day). Rats were fed the ammonium-containing diet starting on day 1 of pregnancy and were maintained on this diet until weaning (at posnatal day 21). After weaning, pups were either fed a normal diet, with no ammonium acetate added, or continued on ammonium until sacrifice.

GLP compliance:

not specified

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 220-260 g

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

After weaning, a group of the pups exposed to ammonia were fed a normal diet without ammonia added (Ammonia -> control group) while others were maintained on the ammonium diet (Ammonium group).

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

No data

Duration of treatment / exposure:

Female rats: From gestational day 1 through lactation day 21.
Pups: 13 days, 1 month or 3 months.

Frequency of treatment:

Daily

Duration of test:

ca. 4 months

Remarks:

Doses / Concentrations:
4293 mg ammonium/kg bw/day (20 % mass of diet)
Basis:
nominal conc.

No. of animals per sex per dose:

For controls: 7 males and 8 females.
For ammonia group: 12 males and 9 females.
For the ammonia -> control group: 8 males and 13 females.

Control animals:

yes

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no data

Details on maternal toxic effects:
No information was provided in the study regarding the health of the dams, but it is likely that the high ammonium dietary concentration made them hyperammonemic. Body weights and food consumption by the dams throughout the study were not reported.

Key result

Dose descriptor:

NOAEL

Effect level:

4 293 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Rats exposed to NH4 + in utero and during lactation (dams received 4293 mg ammonium/kg/day in the diet from gestational day 1 through lactation day 21), which then received a normal diet, had a statistically reduction in body weight gain by 25% and 16% in male and female offspring, respectively, at 120 days of age.
Rats that were continued on the same ammonia diet as their dams had an even greater decrease in body weight gain (27 and 26% for males and females, respectively) at 120 days of age.

Key result

Dose descriptor:

LOAEL

Effect level:

>= 4 293 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

The NOAEL for developmental toxicity was 4293 mg/kg bw/day.

Conclusions:

The NOAEL for developmental toxicity was 4293 mg/kg bw/day.

Executive summary:

Wistar rats were used to study the toxicity effects of prenatal exposure of Ammonium Acetate. For prenatal exposure to ammonia, female rats were made hyperammonemic by feeding them a diet containing Ammonium acetate (4293 mg ammonium/kg bw/day). Rats were fed the ammonium-containing diet starting on day 1 of pregnancy and were maintained on this diet until weaning (at posnatal day 21). After weaning, pups were either fed a normal diet, with no ammonium acetate added, or continued on ammonium until sacrifice.

Rats exposed to NH4 + in utero and during lactation (dams received 4293 mg ammonium/kg/day in the diet from gestational day 1 through lactation day 21), which then received a normal diet, had a statistically reduction in body weight gain by 25 and 16% in male and female offspring, respectively, at 120 days of age.

Rats that were continued on the same ammonia diet as their dams had lower body weight gain (25 and 26% for males and females, respectively) at 120 days of age.

The NOAEL for developmental toxicity was 4293 mg/kg bw/day.

No information was provided in the study regarding the health of the dams, but it is likely that the high ammonium dietary concentration made them hyperammonemic. Body weights and food consumption by the dams throughout the study were not reported.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

4 293 mg/kg bw/day

Additional information

Experimental data on Ammonium Acetate:

In paper, reported by Miñana et al. (1995), Wistar rats were used to study the toxicity effects of prenatal exposure of Ammonium Acetate. For prenatal exposure to ammonia, female rats were made hyperammonemic by feeding them a diet containing Ammonium acetate (4293 mg ammonium/kg bw/day). Rats were fed the ammonium-containing diet starting on day 1 of pregnancy and were maintained on this diet until weaning (at posnatal day 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.

Weight of evidence: Experimental data on Ammonium Acetate and read-across approach from experimental data on analogues Sodium Acetate, Citric Acid, Calcium formate, and Acetic Acid on rats, mice, and rabbits.

Read-across from experimental data on Sodium Acetate:

In the first paper, reported by Kavlock et al., 1987, pregnant CD-1 mice were treated with Sodium Acetate by oral gavage on days 8-12 of gestation at a dose level predicted from a preliminary range finding study to induce a slight degree of maternal toxicity. Day 20 of gestation was considered postnatal day 1 (PD1). On PD1 and 3, the litters were counted and weighed as a unit. No maternal toxicity and no neonatal effects (mortality and body weight) were observed at a dose level of 1000 mg/kg bw/day. By read-across approach, for Ammonium Acetate the NOAEL is calculated to be equal or greater than 939.66 mg/kg bw/day (based on maternal toxicity: mortality, pregnancy and resorption; and on neonatal effects: mortality and body weight).

Read-across from experimental data on Citric Acid:

In the study reported by Wright et al., 1976, a one-generation study is described. This study examines the effect of a 5% dietary supplement of Citric acid (ca. 2.5 g/kg bw/day) on the reproductive capacity of rats and mice. Animals were treated before, during, and after mating. No effects were seen on number of pregnancies, number of young born, or survival of young in treated rats and mice with ca. 2.5 g/kg bw/day Citric Acid, compared to controls. The read-across approach is applied and the NOAEL with Ammonium Acetate is calculated to be 3009.37 mg/kg bw/day on rats and mice (basis for effects: number of pregnancies, number of young born, or survival of young).

Read-across from experimental data on Calcium Formate:

In the third paper, reported by Malorny (1969), a three-generation study was performed with Calcium formate in Wistar rats. Animals were treated by drinking water at doses of0 or 200 mg/kg bw/day of test substance. No effects on fertility were observed. Number, weight and length of offspring did not differ in treated animals from controls. A portion of the offspring was also sacrificed shortly after birth for evaluation of developmental toxicity. No statistical differences in organ or bone abnormalities were found. The growth of treated offspring was similar to controls.The NOAEL for maternal and developmental toxicity was equal or higher than 200 mg/kg bw/day.The read-across approach is applied and the NOAEL for maternal and developmental toxicity with Ammonium Acetate is calculated to be equal or higher than 236.96 mg/kg bw/day (overall effects).

Read-across from experimental data on Acetic Acid:

In the paper reported by FDA (1974), a one-generation study is presented on female mice, rats and rabbits with Acetic acid. Pregnant animals were dosed daily by oral intubation, beginning on day 6 of gestation. Tested doses were 0, 16, 74, 345, and 1600 mg/kg bw/day. No effects on nidation or on maternal or fetal survival were observed at doses up to 1600 mg/kg bw/day. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring in the controls. Based on the experimental results 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 2055.47 mg/kg bw/day for maternal and developmental toxicity in mice, rats, and rabbits.

Justification for classification or non-classification

All these studies have shown no effects on fertility nor on developmental of foetuses, eventhough high doses of chemicals were used, also in a three-generation study with the analogue substance Calcium formate. Based on these negative results, it is considered not necessary to perform more studies on reproduction and developmental toxicity.