Ammonium acetate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in humans

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From June 1983 to October 1986.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study meets generally acceptable scientific standards, valid for assessment.

Principles of method if other than guideline:

Subjects were examined in the Department of Dermatology of Turku University Central Hospital in Finland, between June 1983 and October 1986. Patients were presumed to have prior exposure to citric acid because of its use as a food preservative. During the initial visit, each prospective subject was encouraged to follow an additive-free diet. Histamine chloride (3 mg/mL) and physiological saline were used as positive and negative controls, respectively. The volume of test and control agents administered in the skin prick test was not specified. Tests were read after 15 minutes. 91 patients suffering from chronic or recurrent urticaria or recurrent angioedema were exposed to 2.5 % aqueous solution of citric acid in the skin-prick testing.

GLP compliance:

not specified

Type of study:

other: Skin prick testing

Justification for non-LLNA method:

Data provided from a study with human skin. Data from a publication.

Species:

human

Strain:

other: not applicable

Sex:

male/female

Details on test animals and environmental conditions:

Subjects suffering from chronic or recurrent urticaria or recurrent angioedema of 2 months to 17 yr duration were used in this study.
The subjects were examined in the Department of Dermatology of Turku University Central Hospital between June 1983 and October 1986.

Route:

intradermal

Vehicle:

water

Concentration / amount:

2.5 % aqueous solution of citric acid

Route:

other: no data

Vehicle:

water

Concentration / amount:

2.5 % aqueous solution of citric acid

No. of animals per dose:

91 subjects for the treated group (no data on sex distribution)
247 subjects in the control group (no data on sex distribution)

Details on study design:

- Negative control group: physiological saline.
- Duration of exposure: 15 minutes
- Only weals as large or larger than that of histamine were considered positive. The subject was considered as atopic if they had or had had atopic dermatitis, allergic rhinitis or asthma or had at least one positive skin test reaction with common inhalant allergens.

Positive control substance(s):

yes

Remarks:

(Histamine chloride (3 mg/mL))

Key result

Reading:

1st reading

Hours after challenge:

0.25

Group:

test chemical

Dose level:

2.5%

No. with + reactions:

3

Total no. in group:

91

Clinical observations:

Weals

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 0.25. Group: test group. Dose level: 2.5%. No with. + reactions: 3.0. Total no. in groups: 91.0. Clinical observations: Weals.

Key result

Reading:

rechallenge

Hours after challenge:

0.25

Group:

test chemical

Dose level:

2.5%

No. with + reactions:

0

Total no. in group:

11

Clinical observations:

No effects

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 0.25. Group: test group. Dose level: 2.5%. No with. + reactions: 0.0. Total no. in groups: 11.0. Clinical observations: No effects.

Key result

Reading:

1st reading

Hours after challenge:

0.25

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

247

Clinical observations:

No effects

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 0.25. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 247.0. Clinical observations: No effects.

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

It is concluded that Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.

Executive summary:

In a study of urticarial reactions, 3 subjects of a group of 91 patients suffering from chronic or recurrent urticaria or recurrent angioedema produced positive a skin-prick reaction in response to a 2.5% aqueous solution of citric acid. Upon re-testing, none of them displayed a reproducible positive reaction. None of the 247 normal (non-uticaria) human control subjects displayed a response to citric acid.

Subjects were examined in the Department of Dermatology of Turku University Central Hospital in Finland, between June 1983 and October 1986. Patients were presumed to have prior exposure to citric acid because of its use as a food preservative. During the initial visit, each prospective subject was encouraged to follow an additive-free diet. Histamine chloride (3 mg/mL) and physiological saline were used as positive and negative controls, respectively. The volume of test and control agents administered in the skin prick test was not specified. Tests were read after 15 minutes.

It is concluded that Citric acid (2.5 % aqueous solution) is not sensitizing for the human skin.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in humans

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Documentation insufficient for full assessment. No data on GLP.

Principles of method if other than guideline:

A patch-test was performed on 60 patients with hand eczema, all of whom were involved in handling food with the test substance citric acid. The patients were patch tested with test substance (2.5 % citric acid in petrolatum) using the Finn Chamber method. The exposure lasted for probably 24 hours (covered contact).

GLP compliance:

not specified

Type of study:

patch test

Justification for non-LLNA method:

Patients with hand-eczema were used in this study.

Species:

human

Strain:

other: not applicable

Sex:

male/female

Details on test animals and environmental conditions:

Patients with hand-eczema were used in this study.

Route:

other: epicutaneous, covered contact

Vehicle:

petrolatum

Concentration / amount:

2.5% citric acid in petrolatum.

Concentration / amount:

2.5% citric acid in petrolatum.

No. of animals per dose:

Treated group: 60 patients with hand eczema.

Details on study design:

The patients were patch tested with test substance using the Finn Chamber method.
Duration of exposure: probably 24 h covered contact
Patch test reactions and delayed reactions were evaluated.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5 % citric acid

No. with + reactions:

0

Total no. in group:

60

Clinical observations:

No effects

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2.5 % citric acid. No with. + reactions: 0.0. Total no. in groups: 60.0. Clinical observations: No effects.

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Citric acid was not sensitizing for human skin under test conditions.

Interpretation of results:

GHS criteria not met

Conclusions:

Citric acid was not sensitizing for human skin under test conditions.

Executive summary:

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.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Scientific Review Article. No data on GLP.

Principles of method if other than guideline:

A modified Draize test (species and number of animals not stated) in which the intradermal injection challenge was 3% Glycolic acid and the topical application challenge was 60% Glycolic acid.

GLP compliance:

not specified

Type of study:

Draize test

Justification for non-LLNA method:

A modified Drtaize test.

Species:

other: no data

Strain:

not specified

Sex:

not specified

Route:

other: no data

Vehicle:

no data

Concentration / amount:

Intradermal injection challenge: 3 % Glycolic acid
Topical application challenge: 60 % Glycolic acid

Route:

intradermal and epicutaneous

Vehicle:

no data

Concentration / amount:

Intradermal injection challenge: 3 % Glycolic acid
Topical application challenge: 60 % Glycolic acid

No. of animals per dose:

No data

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

In a modified Draize test (species and number of animals not stated) in which the intradermal injection challenge was 3% and the topical application challenge was 60%, Glycolic Acid was not a sensitizer.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

rechallenge

Group:

positive control

Remarks on result:

not measured/tested

Glycolic acid was not a sensitizer.

Interpretation of results:

GHS criteria not met

Conclusions:

Glycolic acid was not a sensitizer.

Executive summary:

In a modified Draize test (species and number of animals not stated) in which the intradermal injection challenge was 3% and the topical application challenge was 60%, Glycolic Acid was not a sensitizer.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Scientific Review Article. No data on GLP.

Principles of method if other than guideline:

A maximization study using guinea pigs (number of animals not stated) was performed in which induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%.

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A guinea pig test .

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Route:

intradermal and epicutaneous

Vehicle:

no data

Concentration / amount:

Induction: intradermal injection: 10 % Sodium Glycolate
Induction: topical application: 25 % Sodium Glycolate
Challenge: topical application of 25 % Sodium Glycolate

Route:

other: epicutaneous

Vehicle:

no data

Concentration / amount:

Induction: intradermal injection: 10 % Sodium Glycolate
Induction: topical application: 25 % Sodium Glycolate
Challenge: topical application of 25 % Sodium Glycolate

No. of animals per dose:

No data

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

rechallenge

Group:

positive control

Remarks on result:

not measured/tested

Sodium Glycolate was not a sensitizer.

Interpretation of results:

GHS criteria not met

Conclusions:

Sodium Glycolate was not a sensitizer.

Executive summary:

A maximization study using guinea pigs (number of animals not stated) was performed in which induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%.

Sodium Glycolate was not a sensitizer.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Scientific Review Article. No data on GLP.

Principles of method if other than guideline:

A maximization study was performed using guinea pigs (number of animals not stated) in which induction consisted of intradermal injection of 0.2% and topical application of 50% Lactic Acid; challenge consisted of intradermal injection of 0.2% and application of 10%.

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A study with guinea pigs.

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Route:

intradermal and epicutaneous

Vehicle:

no data

Concentration / amount:

Induction: intradermal injection: 0.2 % Lactic acid
Induction: topical application: 50 % Lactic acid
Challenge: intradermal injection: 0.2 % Lactic acid
Challenge: topical application: 10 % Lactic acid

Route:

intradermal and epicutaneous

Vehicle:

no data

Concentration / amount:

Induction: intradermal injection: 0.2 % Lactic acid
Induction: topical application: 50 % Lactic acid
Challenge: intradermal injection: 0.2 % Lactic acid
Challenge: topical application: 10 % Lactic acid

No. of animals per dose:

No data

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

intradermal injection of 0.2% and topical application of 50% Lactic Acid; challenge consisted of intradermal injection of 0.2% and application of 10%.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

rechallenge

Group:

positive control

Remarks on result:

not measured/tested

Lactic Acid was not a sensitizer.

Interpretation of results:

GHS criteria not met

Conclusions:

Lactic Acid was not a sensitizer.

Executive summary:

A maximization study was performed using guinea pigs (number of animals not stated) in which induction consisted of intradermal injection of 0.2% and topical application of 50% Lactic Acid; challenge consisted of intradermal injection of 0.2% and application of 10%.

Lactic Acid was not a sensitizer.

Reference 6

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Scientific Review Article. No data on GLP.

Principles of method if other than guideline:

The sensitization potential of a 12% Ammonium Lactate lotion, pH 5.0-5.5, was examined using 10 guinea pigs. The first induction application consisted of 0.5 mL applications of undiluted material as well as 25 and 50% dilutions. The remaining two induction applications (one per week), as well as the two subsequent challenge applications (applied 2 and 3 weeks after the last induction dose), of 0.5 mL were undiluted lotion. The first induction dose and the two challenge doses were placed under occlusive patches for 24 h; the remaining two induction doses were placed under occlusive patches for 6 h.

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A guinea pig test.

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Route:

epicutaneous, occlusive

Vehicle:

no data

Concentration / amount:

12% Ammonium Lactate lotion (pH 5.0-5.5)

Route:

epicutaneous, occlusive

Vehicle:

no data

Concentration / amount:

12% Ammonium Lactate lotion (pH 5.0-5.5)

No. of animals per dose:

10 guinea pigs (no data on sex distribution)

Details on study design:

The first induction application consisted of 0.5 mL applications of undiluted material as well as 25 and 50% dilutions. The remaining two induction applications (one per week), as well as the two subsequent challenge applications (applied 2 and 3 weeks after the last induction dose), of 0.5 mL were undiluted lotion.
The first induction dose and the two challenge doses were placed under occlusive patches for 24 h; the remaining two induction doses were placed under occlusive patches for 6 h.

Key result

Reading:

1st reading

Group:

test chemical

Dose level:

The first induction application consisted of 0.5ml applications of undiluted material as well as 25 and 50% dilutions

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Key result

Reading:

2nd reading

Group:

test chemical

Dose level:

Two applications one per week of 0.25ml of undiluted

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

two subsequent challenge applications (applied 2 and 3 weeks after the last induction dose), of 0.5 mL were undiluted lotion.

Remarks on result:

no indication of skin sensitisation

One animal was found dead on day 18 (reason not stated). No erythema was observed after induction or challenge applications.

12% Ammonium Lactate lotion was not a sensitizer using guinea pigs.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium Lactate was not a sensitizer to guinea pigs.

Executive summary:

The sensitization potential of a 12% Ammonium Lactate lotion, pH 5.0-5.5, was examined using 10 guinea pigs. The first induction application consisted of 0.5 mL applications of undiluted material as well as 25 and 50% dilutions. The remaining two induction applications (one per week), as well as the two subsequent challenge applications (applied 2 and 3 weeks after the last induction dose), of 0.5 mL were undiluted lotion. The first induction dose and the two challenge doses were placed under occlusive patches for 24 h; the remaining two induction doses were placed under occlusive patches for 6 h.

One animal was found dead on day 18 (reason not stated). No erythema was observed after induction or challenge applications.

12% Ammonium Lactate lotion was not a sensitizer to guinea pigs.

Reference 7

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Scientific Review Article. No data on GLP.

Principles of method if other than guideline:

The "drop-on method" was performed in a test with five guinea pigs.

GLP compliance:

not specified

Type of study:

other: "Drop-on method"

Justification for non-LLNA method:

Test performed with guinea pigs.

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Key result

Reading:

1st reading

Group:

test chemical

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Triacetin was not a sensitizer using guinea pigs.

Interpretation of results:

GHS criteria not met

Conclusions:

Triacetin was not a sensitizer to guinea pigs.

Executive summary:

The "drop-on method" was performed in a test with five guinea pigs.

Triacetin was not a sensitizer.

Reference 8

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Scientific Review Article. No data on GLP.

Principles of method if other than guideline:

The sensitization potential of Triacetin was evaluated in acetone, dioxane, and guinea pig fat (7:2:1) using guinea pigs. The animals were initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks. A vehicle and positive control were used.

GLP compliance:

not specified

Type of study:

not specified

Justification for non-LLNA method:

A guinea pig test.

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Route:

other: no data

Vehicle:

other: acetone, dioxane, and guinea pig fat (7:2:1)

Concentration / amount:

No data

Route:

other: no data

Vehicle:

other: acetone, dioxane, and guinea pig fat (7:2:1)

Concentration / amount:

No data

No. of animals per dose:

No data

Details on study design:

The animals were initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks.
A vehicle and positive control were used.

Key result

Reading:

1st reading

Group:

test chemical

Dose level:

The animals were initially dosed three times over 5 days.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

Animals were rechallenged after 1, 2, 3 weeks.

Remarks on result:

no indication of skin sensitisation

Triacetin was not sensitizing using guinea pigs.

Interpretation of results:

GHS criteria not met

Conclusions:

Triacetin was not sensitizing using guinea pigs.

Executive summary:

The sensitization potential of Triacetin was evaluated in acetone, dioxane, and guinea pig fat (7:2:1) using guinea pigs. The animals were initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks. A vehicle and positive control were used.

Triacetin was not sensitizing.

Reference 9

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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 skin sensitisation 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 skin prick testing on the analogue Citric acid.

GLP compliance:

not specified

Type of study:

other: read-across from a skin prick testing with an analogue

Key result

Reading:

1st reading

Hours after challenge:

0.25

Group:

test chemical

Dose level:

2.5 % citric acid

No. with + reactions:

3

Total no. in group:

91

Clinical observations:

Weals

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 0.25. Group: test group. Dose level: 2.5 % citric acid. No with. + reactions: 3.0. Total no. in groups: 91.0. Clinical observations: Weals.

Key result

Reading:

rechallenge

Hours after challenge:

0.25

Group:

test chemical

Dose level:

2.5 % citric acid

No. with + reactions:

0

Total no. in group:

11

Clinical observations:

No effects

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 0.25. Group: test group. Dose level: 2.5 % citric acid. No with. + reactions: 0.0. Total no. in groups: 11.0. Clinical observations: No effects.

Key result

Reading:

1st reading

Hours after challenge:

0.25

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

247

Clinical observations:

No effects

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 0.25. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 247.0. Clinical observations: No effects.

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Based on the experimental results obtained with the analogue Citric acid (Citric acid, 2.5 % aqueous solution, is not sensitizing for the human skin), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing for the human skin. 

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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for the human skin.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_01 Citric acid) obtained with the analogue Citric acid (Citric acid, 2.5 % aqueous solution, is not sensitizing for the human skin), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing for the human skin.

Reference 10

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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 Citric Acid which shares the same functional group with Ammonium Acetate, also has comparable values for the relevant molecular properties for the skin sensitisation 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 patch-test on the analogue Citric acid.

GLP compliance:

not specified

Type of study:

other: read-across from a patch-test with an analogue

Justification for non-LLNA method:

Patients with hand-eczema were used in the study done with analogue.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5%citric acid (analogue substance)

No. with + reactions:

0

Total no. in group:

60

Clinical observations:

No effects

Remarks on result:

other: Data on analogue substance:1st reading. Hours after challenge: 24.0. Group: test group. Dose level: 2.5 % citric acid. No with. + reactions: 0.0. Total no. in groups: 60.0. Clinical observations: No effects.

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Based on the experimental results obtained with the analogue Citric acid (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), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing for the human skin.

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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for the human skin.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_02 Citric acid) obtained with the analogue Citric acid (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) , the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing for the human skin.

Reference 11

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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 Glycolic Acid (Hidroxyacetic acid) which has a very similar functional group to Ammonium Acetate, also has comparable values for the relevant molecular properties for the skin sensitisation 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 modified Draize test on the analogue Glycolic acid.

GLP compliance:

not specified

Type of study:

other: read-across from a modified Draize test with an analogue

Justification for non-LLNA method:

A modified Drtaize test was performed on analogue.

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

The analogue Glycolic acid was not a sensitizer in a modified Draize test in which the intradermal injection challenge was 3% and the topical application challenge was 60%.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

rechallenge

Group:

positive control

Remarks on result:

not measured/tested

Based on the experimental results obtained with the analogue Glycolic acid (It was not a sensitizer in a modified Draize test in which the intradermal injection challenge was 3% and the topical application challenge was 60%), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

The analogue Glycolic acid (Hidroxyacetic acid) which has a very similar functional group to Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:

- a low log Pow value which is -1.11 for Glycolic acid, and -2.79 for Ammonium acetate,

- a high water solubility which is 1000 g/L at 25 ºC for Glycolic acid, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights which are 76.05 for Glycolic acid, 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 79-14-1	Target chemical 631-61-8
CHEMICAL NAME		Glycolic Acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Experimental data: 79.5 ºC	Experimental data: 114 ºC
Boiling Point		Experimental data: 100 ºC	Estimated data: 312.76 ºC
Density		Experimental data: 1.49 at 25 deg C	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Estimated data: 2.0E-02 mm Hg at 25 ºC	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Experimental data: -1.11	Estimated data: -2.79
Water solubility		Estimated data: 1000 g/L at 25 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 = 164 ppm (Pimephales promelas)   (96 h) LC50 > 5000 mg/L (Brachydanio rerio)	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) EC50 = 141 mg/L (95% confidence interval: 100-200 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:   (72 h) EC50 = 44 mg/L (95% confidence interval: 41.4-46.7 mg/L) (Selenastrum capricornutum)	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 = 1950 mg/kg bw (rat) LD50 = 1920 mg/kg bw (Guinea pig)	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 = 7.7-14 mg/L (rat)	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:   Glycolic acidwas classified as a primary skin irritant when 70% technical glycolic acid, 0.5 mL applied undiluted to abraded and intact skin of one rabbit resulted in primary skin irritation bordering on corrosive. Strong erythema and mild edema were seen on the intact skin and strong erythema and necrosis were seen along the lines of abrasion; these observations were not visible at 72 hr. However, in another study in which the same dose was applied to the intact skin of six rabbits under an occlusive patch for 4 hr and then washed, skin corrosion was not observed at 24 or 48 hr.   Dermal irritation tests using male white rabbits were performed on one face cream product containing 15% glycolic acid and two peeling products containing 25 and 50% glycolic acid;the pH of the products was 4.5. The test dose, 100% of net product, was applied to whole and flaky skin of six rabbits. All test compounds produced some erythema but no edema. The dermic irritation indices were 0.50, 0.33, and 0.38 for the 15, 25, and 50% glycolic acidproducts, respectively, and it was concluded they were "not irritable".	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate, Ammonium Sulphate, and Ammonium Stearate, based on functional group: The substance Ammonium Acetate is considered as not irritating for eyes.
Eye Irritation/Corrosion		Experimental data:   The ocular irritation potential of a number of cosmetic formulations containing glycolic acidwas determined in primary eye irritation studies and the formulations were generally found to be non- or mildly irritating.	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		Experimental data: In a modified Draize test (species and number of animals not stated) in which the intradermal injection challenge was 3% and the topical application challenge was 60%, Glycolic Acid was not a sensitizer.	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 data:   10 groups of 10 male Wistar rats were fed a basal diet or the basal diet with 3% glycolic acidfor 4 weeks to examine glycolic acid’sability to induce calculi formation. Body weights were measured weekly and feed and water consumption were determined during weeks 2 and 4. At necropsy, the urinary tracts were examined grossly and the kidneys, heart, femur, and a section of skeletal muscle were analyzed for oxalate and/or glycolate. All rats appeared normal after 4 weeks. The addition of glycolic acidto basal diet resulted in decreased body weight gain and increased water intake, but it did not affect feed consumption. Glycolic acidwas a potent calculi producer, with deposits being observed in the ureters, urinary bladder, renal tubules, and/or renal pelvis and papilla of all 10 rats. The calculi recovered were composed of calcium oxalate and the calculi from the urinary bladder or renal pelvis were </= 4 mm in diameter. The addition of pyruvate to the diet had a preventive effect on oxalate urolith formation, and at microscopic examination no calcium deposits were found in the kidneys of rats fed pyruvate and pyruvate plus alanine. The kidneys of rats fed glycolic acidhad an average of 2.4% oxalate on a dry weight basis; the addition of pyruvate and alanine reduced oxalate to approximately control values, i.e. 0.2%. No increase in oxalate content was found in the hearts, muscles, or femurs of the glycolate-fed rats. /It was/ found that sodium and potassium pyruvate, and to a lesser extent sodium and potassium bicarbonate and pyruvic acid, did not produce stones in the urinary system. /It was/ reported /in/ similar findings upon addition of magnesium hydroxide, magnesium citrate, and magnesium trisilicate. In both studies the researchers stated that urinary calculi formation was most likely reduced by an increase in urinary citrate concentration, not by decreased oxalate synthesis.   Groups of 10 male Wistar rats were fed a basal diet or the basal diet with 3% glycolic acidfor 3 weeks. Pooled 24-hr urine samples were taken. At dose termination, the animals were necropsied, the kidneys and urinary tracts were examined grossly for calculi, and the kidneys were also analyzed for total oxalate and calcium. The feeding of glycolic acid resulted in a high incidence of oxalate urolithiasis; the uroliths were seen mostly in the kidneys, but some animals also had uroliths in the ureter and urinary bladder. Also, fine crystalline depositions were present throughout the cortex and medulla and clusters of concretions were on the surface or embedded in the renal papilla. The addition of alanine to the diet generally prevented calculi formation. Additionally, the feeding of /a/ diet containing alanine (without glycolic acid)to rats with concretions from previous feeding with glycolic aciddissolved the depositions.     Repeated Dose Toxicity: Inhalation:   Experimental data:   An inhalation study was performed in which rats, 10 per group, were exposed to 0.23, 0.72 or 2.0 mg/L of a 70% glycolic acidsolution for 5 days/week, for 2 weeks; the animals of the 2.0 mL group received only eight exposures due to their deteriorating condition. The animals were observed for 2 weeks after dosing. One animal of the 0.72 mg/L dose group died during the recovery period from dose-related effects. Rats dosed with 2.0 mg/L had increased serum glutamate pyruvate transaminase (SGPT) and serum glutamic-oxaloacetic transaminase (SGOT) values and decreased urine volume and pH. Rats dosed with 0.72 mg/L had increased SGOT values, decreased urine volume, and reversible hepatic effects. No signs of toxicity were observed in rats dosed with 0.23 mg/L. At microscopic examination, hepatic changes were observed in one rat of the 0.23 mg/L group, nine rats of the 0.72 mg/L group, and seven rats of the 2.0 mg/L group. Gross observations included small spleen, liver, and thymus and a distended gastrointestinal tract.     Repeated Dose Toxicity: Dermal:   Experimental data:   Fifty and 70% glycolic acidwere applied to an acetone-cleansed 2 x 2-cm area of the back of two mini-pigs for 15 min. After 8 hr and 7 and 21 days, 4 mm punch biopsies were taken. Epidermal necrosis and some inflammatory infiltrate and dermal necrosis were induced by 70% glycolic acid after 1 day. Some inflammatory infiltrate and dermal growth were observed with 50 and 70% glycolic acidand 12% lactic acid after 7 and 21 days. The depth of wounding of 10, 50, and 70% glycolic acidwas 0.00, 0.202, and 0.464 mm, respectively. The researchers stated glycolic acid “caused disproportionately more collagen staining and deposition at 7 and 21 days compared with the nonspecific reaction measure at 1 day."   Guinea pigs received daily applications (except Sundays) of a 3 mg/sq cm of 5 or 10% glycolic acid, pH 3.0 to two prewashed 8 by 5 cm area of the back for 3 weeks. Application of glycolic acid for 3 weeks produced some erythema and/or flaking of the skin. At microscopic examination, treated skin had a thickening of the epidermis after treatment with 5 and 10% glycolic acid.Application of 5% glycolic acid produced a two-fold increase in the number of epidermal cell layers; no significant difference in the number of cell layers was found for the animals dosed with 5 versus 10% glycolic acid.Up to a four-fold increase in viable epidermal thickness was observed for the glycolic acid-treated skin as compared to the /control/-treated or untreated skin. Hypertrophy of the epithelium lining of the hair follicles of glycolic acid-treated skin was also observed. Although these epidermal changes were observed in glycolic acid treated skin, the barrier integrity of glycolic acid-and control-treated skin was not significantly different.   The present study examined changes in the epidermal basal cell proliferation and the edema response using skin thickness measurements elicited in SKH-1 mice following exposure to simulated solar light (SSL) with or without topical treatment with creams containing glycolic and salicylic acids. The dose of SSL light required to induce measurable edema (MED(BIOL)) in naive, free-moving SKH-1 mice was determined to be 90 mJ. CIE/sq cm. Pretreating the mice with daily (5 days/week) exposures of 14 mJ. CIE/sq cm for 6 weeks resulted in a doubling of the MED(BIOL) to 180 mJ. CIE/sq cm. Topical application of control cream (pH 3.5), or creams containing glycolic acid (10%, pH 3.5) or salicylic acid (4%, pH 3.5) for 6 weeks (5 days/week) increased the MED(BIOL) to 137 mJ. CIE/sq cm. Daily treatments with SSL (14 mJ. CIE/sq cm) and control cream (pH 3.5), glycolic (10%, pH 3.5) or salicylic (4%, pH 3.5) acid-containing creams for 6 weeks (5 days/week) resulted in an MED(BIOL) value of 180 mJ. CIE/sq cm, which was the same as treatment with light alone for 6 weeks. These data indicate that a 6-week treatment of mouse skin with a representative skin cream, with or without representative alpha- and beta-hydroxy acids (glycolic and salicylic acid, respectively), changes the UV light sensitivity; however, treatment with the cream, with or without the acids, does not contribute to the UV sensitivity of mice cotreated with low doses of UV light.   The purpose of this study was to examine the effects of glycolic acidalone or in combination with UVB on skin damage and inflammatory response. ... Guinea pigs were treated with glycolic acid (from 1 to 7 mg/sq cm) alone or in combination with UVB (0.4 or 3 J/sq cm) for 14 days. Skin damage was evaluated by scoring the skin irritation value by the method of Draize and by histopathological observations. Cyclooxygenase 2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production were also assessed. ... Glycolic acid caused an increase in the level of skin damage in a dose- and time-dependent manner. Lower doses (1 and 3 mg/sq cm) of glycolic acid mostly caused erythema and eschar, and these consequently formed scales, whereas higher doses (5 and 7 mg/sq cm) of glycolic acid caused redness, edema and necrotic ulceration. Glycolic acidalso increased the thickness of the epidermal layer, reduced the organization of the stratum corneum and eventually destroyed some parts of the epidermal layer at 7 mg/sq cm. UVB (0.4 and 3 J/sq cm) caused redness and edema as well as reduced the integrity of the stratum corneum. Glycolic acid enhanced the UVB-induced skin damage. The magnitude of the damage caused by combined UVB and glycolic acid treatment was much greater than that caused by glycolic acid or UVB alone. Moreover, partial destruction of the epidermal layer was observed in skin treated with 3 J/sq cm UVB and 3 mg/sq cm glycolic acid.However, glycolic acid did not change the basal and UVB-induced PGE(2) production and COX-2 protein expression.	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:   Negative with and without activation in the Ames test using Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 at 4-5000 ug/plate.	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:   The present study examined the effect of glycolic acidon UV (UVA + UVB)-induced skin tumorigenesis and assessed several significant contributing factors in SKH-1 hairless mice. Inbred hairless female mice (15 animals/group) were irradiated for 5 d/wk at a total dose of 74.85 J/sq cm UVA and 2.44 J/sq cm UVB for 22 wk. Glycolic acidwas applied topically twice a week at a dose of 8 mg/sq cm immediately after UV irradiation. Glycolic acidreduced UV-induced skin tumor development. The protective effect of glycolic acidwas a 20% reduction of skin tumor incidence, a 55% reduction of tumor multiplicity (average number of tumors/mouse), and a 47% decrease in the number of large tumors (larger than 2 mm). Glycolic acidalso delayed the first appearance of tumor formation by about 3 wk. The inhibitory effect of glycolic acid on UV-induced tumor development was accompanied by decreased expression of the following UV-induced cell-cycle regulatory proteins: proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin E, and the associated subunits cyclin-dependent kinase 2 (cdk2) and cdk4. In addition, the expression of p38 kinase, jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase (MEK) also was lower in UV + glycolic acid-treated skin compared with expression in UV-irradiated skin. Moreover, transcription factors activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) activation was significantly lower in UV + glycolic acid-treated skin compared with activation in UV-irradiated skin. These results show that glycolic acidreduced UV-induced skin tumor development. The decreased expression of the cell-cycle regulatory proteins PCNA, cyclin D1, cyclin E, cdk2, and cdk4 and the signal mediators JNK, p38 kinase, and MEK may play a significant role in the inhibitory effect of glycolic acidon UV-induced skin tumor development. In addition, the inhibition of activation of transcription factors AP-1 and NF-kappaB could contribute significantly to the inhibitory effect of glycolic acid.   Male and female albino rats were fed 1 and 2% glycolic acidfor 218-248 days. Decreased growth weight, an increase in renal oxalate, and nephrotoxic effects were observed in the male rats. No effects were observed in female rats or in male rats fed 0.5% glycolic acid.Mortality was 60 and 70% for the 1 and 2% dose groups, respectively, with death beginning at day 89. Four groups of male and female albino rats were fed a 1 % yeast-fortified diet, and glycolic acidwas added to the diet of three of the four groups. The dose groups, which were fed 0.5, 1.0, and 2.0% glycolic acid,consisted of four males and four females, seven males and 11 females, and seven males and five females, respectively. The control group, which was fed untreated feed, consisted of nine males and 11 females. Feed consumption, body weight gains, and signs of toxicity were observed during the study; the kidneys were examined at necropsy and final kidney oxalate content was determined. For the male animals of the 1.0 and 2.0% dose groups, average body weight gains were significantly decreased; the decreased growth rate was consistently noted during the first 91 days of the study. No effect on weight gain was observed for the females. Four of the seven males fed 1.0% and five of the seven males fed 2.0% glycolic aciddied on study, with death being preceded by a marked weight loss over a 2- or 3-week period. No females died on study. The animals that died had granulated, mottled, yellowish brown kidneys and were smaller than those of controls. Microscopic changes were reported for all of the examined kidneys of male rats of the 20% dose group and three of the four males of the 1.0% dose group. The kidneys of the males of the 2% dose group and one of the four males of the 1% dose group had masses of mainly calcium oxalate crystals. No microscopic lesions were reported for male animals of the 0.5% dose group or any of the female animals.	No data
Reproductive Toxicity		Experimental data: A pilot developmental toxicity study was conducted using 70% glycolic acid technical solution in which groups of eight Crl:CD BR gravid rats were dosed by gavage with 125, 250, 500, or 1000 mg/kg of the test material in distilled water at a volume of 10 mL/kg on days 7-21 of gestation. A control group was dosed with vehicle only. Clinical signs were recorded once or twice daily, and observations for morbidity and mortality were also made daily. The dams were weighed on days 1 and 7-22 of gestation. Surviving dams were killed on day 22 of gestation and the fetuses were examined. Maternal toxicity was observed at doses of 500 and 1000 mg/kg. Females of the 500 mg/kg had significant increases in the clinical observations of wet chin and lung noise. For this dose group, body weight changs were significantly reduced between days 21 and 22, but no other significant effects on body weight were observed; feed consumption was not affected at this dose. Abnormal gait and mobility, lung noise, salivation, and stained and wet haircoats were observed for dams on the 1000 mg/kg dose group. Body weight gains were significantly decreased at several intervals; maternal body weights for animals of this dose group were statistically significantly reduced (88% of control) on day 22. Feed consumption was also significantly reduced. One moribund female of the 1000 mg/kg dose group was killed. Ulcerations of the gastric mucosa,distended intestine, and mottled kidneys were observed at necropsy. An in vitro embryo culture study was performed in which rat embryos were removed from the uterus, allowed to develop in culture medium, and on day 10.5 of gestation, groups of 10 embryos were cultured for 46 hours in medium containing 0.5, 2.5, 12.5, 25.0, or 50.0 mM glycolic acid; a control group was also cultured. No effects on embryo development were observed with 0.5 or 2.5 mM glycolic acid.At a concentration of 12.5 mM, crown-rump length, head length, and embryo and visceral yolk sac protein content, somite number, and morphology score were significantly decreased. Structural abnormalities, mainly in the craniofacial region, were observed. Doses greater than 12.5 mM caused embryolethality. This study sought to determine the relative roles of glycolic acid, a toxicologically important metabolite of ethylene glycol, and metabolic acidosis in causing developmental toxicity in Sprague-Dawley rats. To tease apart these two interrelated factors, an experimental approach /was developed/ in which high blood glycolate levels could be achieved, in either the presence or absence of metabolic acidosis. Initially, rats previously implanted with a carotid artery cannula were given, on gestation day 10, 40.3 mmol/kg (2500 mg/kg) of ethylene glycol via gavage, 8.5 mmol/kg (650 mg/kg) of glycolic acidvia gavage, 8.5 mmol/kg (833 mg/kg) of sodium glycolate (pH 7.4) via subcutaneous (sc) injection, or distilled water via gavage (control). Peak serum glycolate was nearly identical (8.4-8.8 mM) in the ethylene glycol, glycolic acid, and sodium glycolate groups and, as expected, ethylene glycol and glycolic acidcaused a metabolic acidosis, but acid base balance was normal with sodium glycolate. Subsequently, these treatments were given on gestation day 6-15 to groups of 25 time-mated rats, followed by fetal evaluation on gestation day 21. Ethylene glycol and glycolic aciddecreased fetal body weights and caused a similar spectrum of developmental effects, including numerous axial skeleton malformations. Sodium glycolate treatment also caused slight decreases in fetal body weight, increases in skeletal variations, and totally malformed fetuses. These results indicate that glycolate, in the absence of metabolic acidosis, can cause the most sensitive of ethylene glycol's developmental effects, whereas metabolic acidosis appears to interact with glycolate at very high doses to markedly enhance teratogenesis. These results support previous studies, which indicated that glycolate is the proximate developmental toxicant for ethylene glycol, and that glycolic acidtoxicokinetic parameters can be used to define a quantitative, physiologically based threshold for ethylene glycol-induced developmental effects. The effects of ethylene glycol and its metabolite, glycolic acid, were compared by culturing day 10.5 rat conceptuses for 46 hr in media containing 0.5, 2.5, 12.5, 25 or 50 mM ethylene glycol or glycolic acid.Ethylene glycol up to 50 mM was essentially without effect, whereas >/= 12.5 mM glycolic acidinhibited embryo growth and development. Craniofacial dysmorphogenesis was observed in 70% of the 12.5 mM glycolic acid embryos (0% in controls). To determine if glycolic acidtoxicity in vitro was an indirect effect of medium acidification, embryos were cultured in 12.5 mM glycolic acid (pH 6.7), 12.5 mM sodium glycolate (pH 7.4), or in control medium (pH 7.4 or 6.7). The percentage of dysmorphic embryos was 67% for the 12.5 mM glycolic acid (pH 6.7) group, 58% for the sodium glycolate (pH 7.4) group, 8% in the pH 6.7 controls, and 0% in the pH 7.4 controls. These results suggest that glycolic acid,not parent ethylene glycol, is the active toxicant for ethylene glycol-induced developmental toxicity and that acidification of culture medium pH plays only a minor role in glycolic acids effects in vitro. The identification of glycolic acid as the active toxicant is important for the risk assessment of ethylene glycol because glycolic acid exhibits dose-rate-dependent, nonlinear kinetics in vivo. Rat whole embryos were exposed for 48 hr (day 9.5-11.5 of gestation) to ethylene glycol (EG) and its metabolites glycolaldehyde (GAl), glycolic acid (GA), glyoxylic acid (GXA), glyoxale (GXAl) and oxalic acid (OXA) at increasing concentrations. Embryotoxic concentrations were achieved within the following range: ethylene glycol (100-200 mM), glycolic acid (3 mM), glyoxal (6 mM), oxalic acid (1-3 mM), glyoxylic acid (0.3-1 mM), glycolaldehyde (0.1-0.2 mM). The pattern of dysmorphogenesis with all compounds including EG showed a general embryotoxicity with diffusely distributed cell necroses with no specific target tissues selectively affected. The results obtained in this study emphasize the hypothesis that the metabolites and not ethylene glycol itself are responsible for the embryotoxicity of ethylene glycol in rats. The developmental toxicity of glycolic acidwas assessed in rats by orally administering solutions of the test material in water over days 7-21 of gestation (the day of copulation plug detection was defined as day 1 of gestation). Groups of 25 mated female Crl:CD BR rats were gavaged at daily dose levels of 0, 75, 150, 300 or 600 mg/kg. The dams were euthanized on day 22 and the offspring were weighed, sexed, and examined for external, visceral, and skeletal alterations. Clear evidence of maternal toxicity was demonstrated at 600 mg/kg; adverse clinical observations were statistically significantly increased (wheezing/lung noise, abnormal gait/staggering, lethargy). In addition, maternal body weights, weight changes, and food consumption were statistically significantly reduced at this dose level. Marginal evidence of maternal toxicity was demonstrated at 300 mg/kg; wheezing/lung noise similar to that seen at 600 mg/kg was observed in 2 of 25 dams. This increase approached statistical significance (p = 0.0553). There was marked evidence of developmental toxicity at 600 mg/kg. Mean fetal weight was statistically significantly reduced while the incidences of skeletal (ribs, vertebra, and sternebra) malformations and variations were statistically significantly increased. At 300 mg/kg/day, there was a slight (2 affected fetuses from 2 litters) increase in the incidence of two skeletal malformations: fused ribs and fused vertebra. Although these increases were not statistically significant (p = 0.0555), they were consistent with findings seen at 600 mg/kg/day and thus were considered relevant. There was no other evidence of developmental toxicity at 300 mg/kg/day nor was any developmental toxicity seen at 150 or 75 mg/kg/day. Thus, the maternal and developmental no-observed-effect level (NOEL) was considered 150 mg/kg. When glycolic acid is given to pregnant rats by mouth on a daily basis, it induces malformations at high, maternally toxic doses. In two studies, there was an 8-9% reduction in fetal body weight and a substantial increase in minor skeletal abnormalities at dose levels associated with mild maternal toxicity. In another study, a marginal increase in fetal abnormalities was seen at a dose associated with marginal maternal toxicity, with no effects on fetal development seen at lower doses. Glycolic acid does not impair fertility or neonatal growth during lactation.	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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for the skin.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_03 Glycolic acid) obtained with the analogue Glycolic acid (It was not a sensitizer in a modified Draize test in which the intradermal injection challenge was 3% and the topical application challenge was 60%), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

Reference 12

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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 Sodium Glycolate (Sodium Hidroxyacetate) which has a very similar functional group to Ammonium Acetate, also has comparable values for the relevant molecular properties for the skin sensitisation 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 Guinea pig maximisation test on the analogue Sodium Glycolate.

GLP compliance:

not specified

Type of study:

other: read-across from a Guinea pig maximisation test with an analogue

Justification for non-LLNA method:

A guinea pig test on the analogue.

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

Data on the analogue: induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

rechallenge

Group:

positive control

Remarks on result:

no indication of skin sensitisation

Based on the experimental results obtained with the analogue Sodium glycolate (It was not a sensitizer in a maximization study using guinea pigs, in which induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

The analogue Sodium glycolate (Sodium hidroxyacetate) which has a very similar functional group to Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:

- a low log Pow value which is -5.19 for Sodium glycolate, and -2.79 for Ammonium acetate,

- a high water solubility which is 1000 g/L at 25 ºC for Sodium glycolate, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights which are 98.03 for Sodium glycolate, 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 2836-32-0	Target chemical 631-61-8
CHEMICAL NAME		Sodium glycolate	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		Estimated data: 4.58E-10 mm Hg at 25 ºC	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Estimated data: -5.19	Estimated data: -2.79
Water solubility		Estimated data: 1000 g/L at 25 ºC	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 = 6700 mg/kg bw (mouse) LD 50 = 7110 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		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		Experimental results: A maximization study using guinea pigs (number of animals not stated) was performed in which induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%. Sodium Glycolate was not a sensitizer.	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		No data	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	No data	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: 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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for guinea pigs.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_04 Sodium glycolate) obtained with the analogue Sodium glycolate (It was not a sensitizer in a maximization study using guinea pigs, in which induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

Reference 13

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in vivo

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 Lactic Acid which has a very similar functional group to Ammonium Acetate, also has comparable values for the relevant molecular properties for the skin sensitisation 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 Guinea pig maximisation test on the analogue Lactic acid.

GLP compliance:

not specified

Type of study:

other: read-across from a Guinea pig maximisation test with an analogue

Justification for non-LLNA method:

A guinea pig test was performed on analogue substance.

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

Induction consisted of intradermal injection of 0.2% and topical application of 50%of the analogue Lactic Acid

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

rechallenge

Group:

positive control

Remarks on result:

not measured/tested

Based on the experimental results obtained with the analogue Lactic acid (It was not a sensitizer in a maximization study using guinea pigs in which induction consisted of intradermal injection of 0.2% and topical application of 50% Lactic Acid; challenge consisted of intradermal injection of 0.2% and application of 10%), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

The analogue Lactic acid which has a very similar functional group to Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:

- a low log Pow value which is -0.72 for Lactic acid, and -2.79 for Ammonium acetate,

- a high water solubility which is 1000 g/L for Lactic acid, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights which are 90.08 for Lactic acid, 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. Data Matrix , Analogue Approach.

CAS Number		Source chemical 50-21-5	Target chemical 631-61-8
CHEMICAL NAME		Lactic acid	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Experimental data: 16.8 ºC	Experimental data: 114 ºC
Boiling Point		Calculated data: 204.2ºC	Estimated data: 312.76 ºC
Density		Measured data: 1.26 g/mL	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Experimental data: 0.0813 mm Hg at 25 ºC	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Experimental data: -0.72	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		Experimental data: Easily 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		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: LD50= 4875mg/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: LC50=7.94mg/L (rat)	No data
Acute Toxicity: Dermal		Experimental data: LD50>2000mg/kg bw (rabbit)	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:   A 5% aqueous solution of Lactic Acid, 0.2 mL, was "very slightly irritant" after repeated application to shaved rat skin (number of animals not stated). One-half milliliter of 5 and 10% aqueous solution Lactic Acidwas applied for 4 hr to the clipped dorsum of rabbits (number and sex not stated) using occlusive patches; the treatment sites had been prehydrated for 60 min immediately prior to dosing. The 5% solution was "virtually nonirritant," and the 10% solution was "only slightly irritant, causing similar effects to those of marketed skin care creams."   Two milliliters/animal of the test substance as supplied (100%) and in dilution (10 and 20%) were applied to the right and left flanks of each of three rabbits. Daily readings were expressed as a weekly average. Qualitative evaluation was made for thickening and dryness of the skin, and microscopic examinations were made after 6 weeks of dosing. Recovery from cutaneous injury was determined by examining the skin 7 days after the last application. Undiluted Lactic Acidproduced severe orthoergic intolerance and dosing was discontinued after 1 week of treatment. Both 10 and 20% Lactic Acidwere well tolerated, with mean maximum irritation indices (MMII) of 0.50 and 1.00, respectively.	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:   Applied to rabbit eyes, reaction at 24 hours has been graded 8 on scale of 1 to 10. If allowed to remain for 60 seconds ... both full strength acid and 50% solution in water caused persistent scarring.   On albino guinea pig eyes 30% Lactic Acidhas produced slight corneal clouding with long-lasting hyperemia and thickening of lids; 5% and 10% solution caused hyperemia of conjunctiva and lids lasting only 5 to 24 hours.   Full strength acid was applied to rabbit's cornea without subsequent irrigation and within 4 days necrosis and loss of superficial layers of cornea in anterior chamber was hypopyon and adhesion of iris to cornea in periphery corneal endothelium.	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		Experimental results: A maximization study was performed using guinea pigs (number of animals not stated) in which induction consisted of intradermal injection of 0.2% and topical application of 50% Lactic Acid; challenge consisted of intradermal injection of 0.2% and application of 10%. Lactic Acid was not a sensitizer.	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: LOAEL = 880 mg/kg bw	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	Negative	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		Negative	No data
Reproductive Toxicity		TOXICITY TO REPRODUCTION: No data DEVELOPMENTAL TOXICITY / TERATOGENICITY: NOAEL: >= 570 (mg/kg-bw/day)	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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for guinea pigs.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_05 Lactic acid) obtained with the analogue Lactic acid (It was not a sensitizer in a maximization study using guinea pigs in which induction consisted of intradermal injection of 0.2% and topical application of 50% Lactic Acid; challenge consisted of intradermal injection of 0.2% and application of 10%), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

Reference 14

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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 Ammonium Lactate which has a very similar functional group to Ammonium Acetate, also has comparable values for the relevant molecular properties for the skin sensitisation 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 Guinea pig maximisation test on the analogue Ammonium lactate.

GLP compliance:

not specified

Type of study:

other: read-across from a Guinea pig maximisation test with an analogue

Justification for non-LLNA method:

A guinea pig study performed on analogue

Key result

Reading:

1st reading

Dose level:

Data on analogue: 0.5ml applications of undiluted material as well as 25 and 50% dilutions

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Key result

Reading:

2nd reading

Group:

test chemical

Dose level:

Data on the analogue: two applications one per week of 0.25ml of undiluted

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

Data on the analogue: two subsequent challenge applications (applied 2 and 3 weeks after the last induction dose), of 0.5 mL were undiluted lotion

Remarks on result:

no indication of skin sensitisation

Based on the experimental results obtained with the analogue Ammonium lactate (It was not a sensitizer using guinea pigs, because no erythema was observed after induction or challenge applications), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

The analogue Ammonium lactate which has a very similar functional group to Ammonium acetate, also has comparable values for the relevant molecular properties. These properties are:

- a low log Pow value which is -3.84 for Ammonium lactate, and -2.79 for Ammonium acetate,

- a high water solubility which is 2247 g/L for Ammonium lactate, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights which are 107.06 for Ammonium lactate, 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 515-98-0	Target chemical 631-61-8
CHEMICAL NAME		Ammonium lactate	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Experimental data: 16.8 ºC	Experimental data: 114 ºC
Boiling Point		Calculated data: 204.2ºC	Estimated data: 312.76 ºC
Density		Measured data: 1.26 g/mL	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Experimental data: 0.0813 mm Hg at 25 ºC	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Experimental data: -0.72	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		Easily 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		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:   LD50= 180mg/kg bw (rat/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		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		Experimental results:   Ammonium lactate (12 %) was not irritating for guinea pigs.	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 results:   Ammonium lactate (12 %) was irritating for eyes.	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		Experimental results: The sensitization potential of a 12% Ammonium Lactate lotion, pH 5.0-5.5, was examined using 10 guinea pigs. The first induction application consisted of 0.5 mL applications of undiluted material as well as 25 and 50% dilutions. The remaining two induction applications (one per week), as well as the two subsequent challenge applications (applied 2 and 3 weeks after the last induction dose), of 0.5 mL were undiluted lotion. The first induction dose and the two challenge doses were placed under occlusive patches for 24 h; the remaining two induction doses were placed under occlusive patches for 6 h. One animal was found dead on day 18 (reason not stated). No erythema was observed after induction or challenge applications. 12% Ammonium Lactate lotion was not a sensitizer to guinea pigs.	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		No data	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	Negative	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	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: 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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for guinea pigs.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_06 Ammonium lactate) obtained with the analogue Ammonium lactate (It was not a sensitizer using guinea pigs, because no erythema was observed after induction or challenge applications), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

Reference 15

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in vivo

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: see 'Remark'

Remarks:

The analogue Triacetin (triester of glycerin and acetic acid) is quickly metabolized to glycerol and acetic acid, which shares the functional group with Ammonium Acetate, and also has comparable values for the relevant molecular properties for the skin sensitisation 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 "Drop-on method" on the analogue Triacetin.

GLP compliance:

not specified

Type of study:

other: read-across from a "Drop-on method" with an analogue

Justification for non-LLNA method:

Study performed with guinea pigs.

Key result

Reading:

1st reading

Group:

test chemical

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Based on the experimental results obtained with the analogue Triacetin (It was not a sensitizer in the "drop-on method" with five guinea pigs), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

The analogue Triacetin (triester of glycerin and acetic acid) is quickly metabolized to glycerol and acetic acid, which shares the functional group with AmmoniumAcetate, and also has comparable values for the relevant molecular properties. These properties are:

- a low log Pow value which is 0.25 for Triacetin, and -2.79 for Ammonium acetate,

- a high water solubility which is 58 g/L for Triacetin at 25 ºC, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights which are 218.21 for Triacetin, 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 102-76-1	Target chemical 631-61-8
CHEMICAL NAME		Triacetin	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Experimental data: 78 ºC	Experimental data: 114 ºC
Boiling Point		Experimental data: 259 ºC	Estimated data: 312.76 ºC
Density		Experimental data: 1.1562 at 25 ºC/4 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Experimental data: 0.00248 mm Hg at 25 ºC	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Experimental data: 0.25	Estimated data: -2.79
Water solubility		Experimental data: 5.80 E+04 mg/L at 25 ºC	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental data: 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:   (48 h) LC50 = 174 mg/L (Cyprinus carpio)   (48 h) LC50 = 170 mg/L (Leuciscus idus)	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:   LD50= 1100 mg/kg bw (mouse) LD50= 3000 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: (4 h) LC50 >1.721 mg/L (Rat)	No data
Acute Toxicity: Dermal		Experimental data:   LD50 >20 mL/kg bw (Guinea pig)   LD50 >5 g/kg bw (Rabbit)	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: Groups of three, three, and two guinea pigs were used to determine the irritation potential of Triacetin. At the end of a 14-day observation period, it was, at most, slightly irritating to skin. However, in one study, erythema, slight edema, alopecia, and desquamation were observed. Investigators examined/ the dermal irritation potential of Triacetin in guinea pigs. Doses of 5 and 10 cc/kg were applied under an occlusive patch for 24 hr. Slight erythema was observed. Occlusive patches with 5 to 20 cc/kg bw were tested using three animals. Slight edema and "1-3 erythema" were observed. Slight skin irritation was observed in the high dose animals.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate, Ammonium Sulphate, and Ammonium Stearate, based on functional group: The substance Ammonium Acetate is considered as not irritating for eyes.
Eye Irritation/Corrosion		Experimental data: In a study using groups of four rabbits, in which ocular tissue sampling was done, 0.1 mL was instilled into both eyes. A control group of four rabbits was not treated. Animals were killed after 2 or 24 hr. In the Draize test, Triacetin had a total Draize score of 1 after 2 hr (0 for the cornea and 0.7 for the conjunctiva); corneal thickness did not change. After 2 hr, the corneal percentage of dry weight/wet weight was significantly decreased, but not after 24 hr, compared to controls. No difference was observed for conjunctival percentage of dry weight/wet weight or Evan's blue concentration/dry weight at 2 or 24 hr. When placed (0.1 mL) undiluted into rabbit eyes, Triacetin was slightly to moderately irritating. It was not irritating when immediately irrigated for 6 min.	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		Experimental results:  The "drop-on method" was performed in a test with five guinea pigs. Triacetin was not a sensitizer. The sensitization potential of Triacetin was evaluated in acetone, dioxane, and guinea pig fat (7:2:1) using guinea pigs. The animals were initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks. A vehicle and positive control were used. Triacetin was not sensitizing.	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:   In short-term feeding studies, Triacetin affected weight gain. Triacetin was not toxic in short-term studies when administered via inhalation or parenterally or in subchronic studies when administered via feed or inhalation.   In the rat, inhalation of 250 ppm Triacetin (6 hr/day, 5 days/wk for 13 weeks) or saturated triacetin vapor (6 hr/day for 5 days) produced no clinical signs of toxicity or histopathological effects.   In 3-month feeding studies, growing rats were found to tolerate up to 20% triacetin in the diet, but diets containing 60% triacetin caused marked retardation of body weight gain and considerable increase in deaths.   Groups of eight male Sprague-Dawley rats that were fed diets containing 30% triacetin (and 30% glycerin or propylene glycol) as a starch substitute for 3 to 4 or 12 to 13 weeks, growth was relatively poor. Liver enlargement was observed in all animals.   The metabolic effect of Triacetin on intestinal mucosal cells and plasma substrates in male Sprague-Dawley rats was assessed /in a sub-chronic study. Rats were fed for 30 days a diet in which 28.5% of the total calories were supplied by Triacetin. No overt signs of toxicity were observed.   Investigators exposed a group of three rats to 250 ppm triacetin by inhalation for 6 hr per day, 5 days per week, for 64 days. The no-observed-effect level (NOEL) was 250 ppm. Three rats were also exposed to 8271 ppm triacetin for 6 hr per day for 64 days. The NOEL was 8271 ppm.   Groups of three, three, and two guinea pigs were used to determine the irritation potential of triacetin. At the end of a 14-day observation period, erythema, slight edema, alopecia, and desquamation were observed.	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:   Negative in the Ames test using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 at concentrations of 50, 150, 500, 1500, 5000 ug/plate with and without metabolic activation.   The mutagenic potential of Triacetin was evaluated in a suspension test with and without metabolic activation. Test concentrations were 0.0013%, 0.00065%, and 0.000325% with S. typhimurium strains TA1535, TA1537, and TA1538 and 1.25%, 2.5%, and 5.0% with Saccharomyces cerevisiae strain D4. DMSO was used as the solvent. Appropriate negative and positive controls were used and gave expected results. Triacetin was not mutagenic in the suspension tests with or 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: 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 (body weight decreased by 16-27%).   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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for guinea pigs.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_07 Triacetin) obtained with the analogue Triacetin (It was not a sensitizer in the "drop-on method" with five guinea pigs), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

Reference 16

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in vivo

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: see 'Remark'

Remarks:

The analogue Triacetin (triester of glycerin and acetic acid) is quickly metabolized to glycerol and acetic acid, which shares the functional group with Ammonium Acetate, and also has comparable values for the relevant molecular properties for the skin sensitisation endpoint.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See attached reporting format.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from published experimental data from a non-standard method on the analogue Triacetin.

GLP compliance:

not specified

Type of study:

other: read-across from a non-standard method with an analogue

Justification for non-LLNA method:

A guinea pig test was performed with the analogue.

Key result

Reading:

1st reading

Group:

test chemical

Dose level:

Data on the analogue: the animals were initially dosed three times over 5 days.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Group:

test chemical

Dose level:

Data on the analogue: animals were rechallenged after 1, 2, 3 weeks.

Remarks on result:

no indication of skin sensitisation

Based on the experimental results obtained with the analogue Triacetin (It was not a sensitizer in guinea pigs initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

The analogue Triacetin (triester of glycerin and acetic acid) is quickly metabolized to glycerol and acetic acid, which shares the functional group with AmmoniumAcetate, and also has comparable values for the relevant molecular properties. These properties are:

- a low log Pow value which is 0.25 for Triacetin, and -2.79 for Ammonium acetate,

- a high water solubility which is 58 g/L for Triacetin at 25 ºC, and 1480 g/L at 4 ºC for Ammonium acetate, and

- similar molecular weights which are 218.21 for Triacetin, 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 102-76-1	Target chemical 631-61-8
CHEMICAL NAME		Triacetin	Ammonium acetate
PHYSICO-CHEMICAL DATA
Melting Point		Experimental data: 78 ºC	Experimental data: 114 ºC
Boiling Point		Experimental data: 259 ºC	Estimated data: 312.76 ºC
Density		Experimental data: 1.1562 at 25 ºC/4 ºC	Experimental results: 1.07-1.17 g/cm3 at 20 ºC
Vapour Pressure		Experimental data: 0.00248 mm Hg at 25 ºC	Estimated data: 0.02 Pa at 25 ºC
Partition Coefficient (log Kow)		Experimental data: 0.25	Estimated data: -2.79
Water solubility		Experimental data: 5.80 E+04 mg/L at 25 ºC	Experimental results: 1480 g/L at 4 ºC
ENVIRONMENTAL FATE and PATHWAY
Aerobic Biodegradation		Experimental data: 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:   (48 h) LC50 = 174 mg/L (Cyprinus carpio)   (48 h) LC50 = 170 mg/L (Leuciscus idus)	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:   LD50= 1100 mg/kg bw (mouse) LD50= 3000 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: (4 h) LC50 >1.721 mg/L (Rat)	No data
Acute Toxicity: Dermal		Experimental data:   LD50 >20 mL/kg bw (Guinea pig)   LD50 >5 g/kg bw (Rabbit)	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: Groups of three, three, and two guinea pigs were used to determine the irritation potential of Triacetin. At the end of a 14-day observation period, it was, at most, slightly irritating to skin. However, in one study, erythema, slight edema, alopecia, and desquamation were observed. Investigators examined/ the dermal irritation potential of Triacetin in guinea pigs. Doses of 5 and 10 cc/kg were applied under an occlusive patch for 24 hr. Slight erythema was observed. Occlusive patches with 5 to 20 cc/kg bw were tested using three animals. Slight edema and "1-3 erythema" were observed. Slight skin irritation was observed in the high dose animals.	Weight of evidence: Read-across approach from experimental data on analogues Potassium Acetate, Ammonium Sulphate, and Ammonium Stearate, based on functional group: The substance Ammonium Acetate is considered as not irritating for eyes.
Eye Irritation/Corrosion		Experimental data: In a study using groups of four rabbits, in which ocular tissue sampling was done, 0.1 mL was instilled into both eyes. A control group of four rabbits was not treated. Animals were killed after 2 or 24 hr. In the Draize test, Triacetin had a total Draize score of 1 after 2 hr (0 for the cornea and 0.7 for the conjunctiva); corneal thickness did not change. After 2 hr, the corneal percentage of dry weight/wet weight was significantly decreased, but not after 24 hr, compared to controls. No difference was observed for conjunctival percentage of dry weight/wet weight or Evan's blue concentration/dry weight at 2 or 24 hr. When placed (0.1 mL) undiluted into rabbit eyes, Triacetin was slightly to moderately irritating. It was not irritating when immediately irrigated for 6 min.	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		Experimental results:  The "drop-on method" was performed in a test with five guinea pigs. Triacetin was not a sensitizer. The sensitization potential of Triacetin was evaluated in acetone, dioxane, and guinea pig fat (7:2:1) using guinea pigs. The animals were initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks. A vehicle and positive control were used. Triacetin was not sensitizing.	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:   In short-term feeding studies, Triacetin affected weight gain. Triacetin was not toxic in short-term studies when administered via inhalation or parenterally or in subchronic studies when administered via feed or inhalation.   In the rat, inhalation of 250 ppm Triacetin (6 hr/day, 5 days/wk for 13 weeks) or saturated triacetin vapor (6 hr/day for 5 days) produced no clinical signs of toxicity or histopathological effects.   In 3-month feeding studies, growing rats were found to tolerate up to 20% triacetin in the diet, but diets containing 60% triacetin caused marked retardation of body weight gain and considerable increase in deaths.   Groups of eight male Sprague-Dawley rats that were fed diets containing 30% triacetin (and 30% glycerin or propylene glycol) as a starch substitute for 3 to 4 or 12 to 13 weeks, growth was relatively poor. Liver enlargement was observed in all animals.   The metabolic effect of Triacetin on intestinal mucosal cells and plasma substrates in male Sprague-Dawley rats was assessed /in a sub-chronic study. Rats were fed for 30 days a diet in which 28.5% of the total calories were supplied by Triacetin. No overt signs of toxicity were observed.   Investigators exposed a group of three rats to 250 ppm triacetin by inhalation for 6 hr per day, 5 days per week, for 64 days. The no-observed-effect level (NOEL) was 250 ppm. Three rats were also exposed to 8271 ppm triacetin for 6 hr per day for 64 days. The NOEL was 8271 ppm.   Groups of three, three, and two guinea pigs were used to determine the irritation potential of triacetin. At the end of a 14-day observation period, erythema, slight edema, alopecia, and desquamation were observed.	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:   Negative in the Ames test using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 at concentrations of 50, 150, 500, 1500, 5000 ug/plate with and without metabolic activation.   The mutagenic potential of Triacetin was evaluated in a suspension test with and without metabolic activation. Test concentrations were 0.0013%, 0.00065%, and 0.000325% with S. typhimurium strains TA1535, TA1537, and TA1538 and 1.25%, 2.5%, and 5.0% with Saccharomyces cerevisiae strain D4. DMSO was used as the solvent. Appropriate negative and positive controls were used and gave expected results. Triacetin was not mutagenic in the suspension tests with or 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: 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 (body weight decreased by 16-27%).   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.

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium acetate is considered to be not sensitizing for guinea pigs.

Executive summary:

Based on the experimental results (reported under the endpoint record 07.04.01_08 Triacetin) obtained with the analogue Triacetin (It was not a sensitizer in guinea pigs initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks), the read-across approach was applied and the substance Ammonium acetate is considered to be also not sensitizing.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Weight of evidence: Ammonium Acetate is considered as not sensitising.

Read-across approach from experimental data on Citric acid:

Citric acid, 2.5 % aqueous solution, is not sensitizing for the human skin. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing for 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. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing for human skin.

Read-across approach from experimental data on Glycolic acid:

It was not a sensitizer in a modified Draize test in which the intradermal injection challenge was 3% and the topical application challenge was 60%. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing.

Read-across approach from experimental data on Sodium glycolate:

It was not a sensitizer in a maximization study using guinea pigs, in which induction consisted of intradermal injection of 10% and topical application of 25% Sodium Glycolate; the challenge application was 25%. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing.

Read-across approach from experimental data on Lactic acid:

It was not a sensitizer in a maximization study using guinea pigs in which induction consisted of intradermal injection of 0.2% and topical application of 50% Lactic Acid; challenge consisted of intradermal injection of 0.2% and application of 10%. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing.

Read-across approach from experimental data on Ammonium lactate:

It was not a sensitizer using guinea pigs, because no erythema was observed after induction or challenge applications. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing.

Read-across approach from experimental data on Triacetin:

It was not a sensitizer in the "drop-on method" with five guinea pigs. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing.

It was not a sensitizer in guinea pigs initially dosed three times over 5 days and challenged after 1, 2, or 3 weeks. Based on these results, the read-across approach was applied and Ammonium acetate is considered to be also not sensitizing.

Short description of key information:

Skin sensitisation: Weight of evidence: Ammonium acetate is considered to be not sensitizing. Read-across approach from experimental results on Citric Acid, Glycolic Acid, Sodium Glycolate, Lactic Acid, Ammonium Lactate, and Triacetin.

Justification for classification or non-classification

Skin sensitisation: Not sensitising.