Ammonium chloride

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In aqueous solution, ammonium salts are completely dissociated into NH4+ and a corresponding anion. This equilibrium depends on temperature, pH and ionic strength of the water in the environment. Un-ionized NH3 species exists in the aquatic environments and the fraction (NH3/(NH3 +NH4+)) steeply increases with elevated pH value or temperature. It is well known that toxicity to aquatic organisms has been attributed to un-ionized ammonia (NH3) species, and NH4 + species is considered to be non- or significantly less-toxic (Emerson et al., 1975). However, recent developments in assessing ammonia toxicity clearly show that in contrast to earlier assumptions where un-ionized ammonia was considered to be the toxic component, both the uncharged and charged molecule are toxic. Therefore, a joint toxicity model has been proposed, with ammonia causing most toxicity at high pH values and ammonium ion also contributing to toxicity at lower pH values (U.S. EPA 1999, OECD 2007).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance: diammonium sulfate, CAS 7783-20-2
Target substance: ammonium chloride, CAS 12125-02-9

3. ANALOGUE APPROACH JUSTIFICATION
It is generally accepted, that the principal toxic component of ammonium salts such as ammonium chloride or -sulphate is ammonia, rather than the corresponding anion (see also: OECD 2004, SIDS ammonium chloride or OECD 2007 ammonium sulphate). Therefore toxicity values for ammonium salts (such as: ammonium -sulphates, phosphates, carbonates, chlorides or nitrates), where the major toxic component is ammonia, can be considered as equivalent. Consequently, this hazard assessment comprises the total topic of ammonia toxicity.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test was carried out according to Amdur MO and Mead J (1958). Mechanics of respiration in unanesthetized guinea pigs. Am J Physiol. 192: 364-368.
Four salts were tested: ammonium sulfate, ammonium bisulfate, sodium sulfate, copper sulfate.

GLP compliance:

not specified

Test type:

other: Amdur and Mead (1958). Mechanics of respiration in unanesthetized guinea pigs. Amer. J.Physiol., 192, 364-368.

Limit test:

no

Species:

guinea pig

Strain:

other:

Remarks:

Random-bred guinea pigs

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 200 - 300 g

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

not specified

Vehicle:

other: no data

Details on inhalation exposure:

The aerosols of the test substance were generated with a Dautrebande D80 aerosol generator. These generators produce it heterogeneous aerosol in the submicrometer size range. The particle size can be varied by varying the concentration of the solution in the nebulizer.
For measurement of particle size, the aerosol was collected directly on an electron microscope grid by electrostatic precipitation. The particles were sized an electron photomicrograph. The count median diameter, the geometric standard deviation, and the mass median diameter (MMD) were calculated.
For measurement of concentration, a sample of the aerosol was collected on a membrane filter. The sample was then dissolved in demineralized water. The ammonium sulfate was measured with an ion electrode or by increase in conductivity.
The main airstream entering the exposure chamber was filtered to remove extraneous particles and dried. The relative humidity of the exposμre chamber atmosphere was 50 %.
The mass median diameter (MMD) was 0.1 - 0.8 µm.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

1 h

Concentrations:

0.13, 0.20 0.30, 0.81 mg/m³

No. of animals per sex per dose:

10 per group

Control animals:

other: pre-exposure values

Statistics:

Student's paired t-test

The effects of the four sulfate salts on resistance and compliance are presented in Table 1 (see attached file).

Ammonium sulfate caused a statistically significant decrease in compliance at all the concentrations and particle sizes tested. Two of the exposures produced an increase in resistance and two did not. According to the authors, the resistance increase produced by the 0.3 µm particles at a concentration of 1 mg/m³ was in good agreement with values found for a smaller group of animals in a previous study (data not shown). Except for the fact that there was a minimal response to the 0.2 -µm particles, the response per microgram of sulfate was greater as the particle size decreased.

Interpretation of results:

study cannot be used for classification

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

other information

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Principles of method if other than guideline:

no data

GLP compliance:

not specified

Test type:

other: no data

Limit test:

no

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

no data

Route of administration:

inhalation: aerosol

Vehicle:

other: water

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of conditioning air: Ammonium sulfate aerosol was generated from an aqueous solution with either one or two Retec nebulizers (Retec Development Laboratory, Portland, Oregon) and dried by mixing with dry air and passing it through a heated glass tube. Two nebulizers were used at concentrations greater than 500 mg/m³.
- System of generating particulates/aerosols: Retec nebulizers (Retec Development Laboratory, Portland, Oregon)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

8 h

Concentrations:

500 - 600, 600 - 700, 800 - 900 mg/m³

No. of animals per sex per dose:

6 - 20

Control animals:

not specified

Mortality:

Mortality rates:
500 - 600 mg/m³: 0/6
600 - 700 mg/m³: 1/6
800 - 900 mg/m³: 8/20
The animals dying during exposure appeared to do so as a result of acute shock and airway constriction. Any sudden noise or other disturbance was likely to precipitate such an event. After exposure, the survivors recovered without any noticable effect.

Interpretation of results:

study cannot be used for classification

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 433 draft (Acute Inhalation Toxicity: Fixed Concentration Procedure) (not officially approved)

Deviations:

not specified

GLP compliance:

not specified

Test type:

other: effect on respiratory defense system

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hilltop Lab Animals, Inc. (Chatsworth, Calif., USA)
- Weight at study initiation: ca. 200 g

ENVIRONMENTAL CONDITIONS
Both temperature and humidity were controlled in this study. Air was supplied to the chambers at about 0.1 - 0.3 m³/min.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: water

Mass median aerodynamic diameter (MMAD):

ca. 0.4 µm

Details on inhalation exposure:

Rats were exposed by inhalation to radioactive tracer particles and then randomly divided into experimental and control groups. One hour after a 20-min nose-only exposure to tracer particles, animals were placed in individual compartments in open-mesh stainless steel exposure cages. Cages were placed on one level only of a 1 m³ stainless steel chamber for a 4-h exposure to either purified or intentionally polluted air.

The tracer particles, tagged with tightly bound 51Cr, were monodisperse polystyrene latex (PSL) spheres having geometric diameters near 1.4 µm. Aerosols were produced from an aqueous suspension of 0.1% solids (by volume), using a Lovelace-type laboratory compressed-air nebulizer. The MMAD of the aerosol particles, as determined with a multistage laboratory impactor was about 1.6 µm.

Tracer particles were aerosolized, dried, brought to charge equilibrium, and passed into a nose-only exposure chamber. The individual tubes for holding rats in the device were made of perforated metal and were thin-walled to reduce thermal stress due to body heat.

The average amount of tracer material deposited per rat was less than 0.1 µCi, which is contained in less than 1 µg of particles. After the deposition of tracer particles was completed, the rats' noses were washed with water to remove radioactive particles. The animals were then placed in individual plastic counting tubes and inserted in a collimated counting apparatus. All rats that underwent deposition of PSL particles were counted twice for 100 s in this apparatus before they were placed in the pollutant exposure chambers. When the 4-h clean air or pollutant exposure was completed, the animals were periodically put into individual plastic counting tubes and the amount of radioactivity in the respiratory tract was determined at five additional predetermined times for up to 17 d. Fecal samples were collected from each rat 11 times during the first 48 h after tracer particle deposition. Coprophagy was minimized by the use of 1/2-inch mesh wire cage bottoms and by the frequent fecal collections.

Clearance curves were determined for each animal and half times obtained from least-squares fits for short- and long-term clearance data. Group mean values for pollutant-exposed and sham-exposed (control) groups were calculated. Half-times for experimental groups were subtracted from those for control groups and the differences tested for significance at the 90% level, using a two-tailed t-test.

Purified air supplied to the exposure chambers had been passed successively through a coarse particulate filter, a humidifier, a heater, and a high-efficiency particulate (HEPA) filter. Both temperature and humidity were controlled in this system. Air was supplied to the chambers at about 0.1-0.3 m³/min. Ammonia levels, due to the presence of rats, were measured as about 0.25 ppm or less under these conditions of exposure.

Stable, controllable salt aerosols with MMAD between 0.4 and 0.6 µm and sulfuric acid aerosols with MMAD of 1.0 µm, at mass concentrations up to 3 or 4 mg/m³ in air, were generated with compressed-air nebulizers loaded with aqueous sulfate solutions. A Collison-type three-jet nebulizer followed by a 85 Kr charge neutralizer and air-dilution drier, was used for ammonium sulfate and ferric sulfate particle generation. Under low humidity (30-40%) chamber conditions these aerosols were dry and were sized by electron microscopy. Ferric sulfate and ammonium sulfate aerosols were collected on electron microscope grids, using an electrostatic precipitator. The size distribution, count median diameter, mass median diameter, and geometric standard deviation were then determined by analysis of photographs. At high humidity (greater than 80%) the aerosols were wet and the multistage laboratory impactor was used.

Sulfuric acid aerosols were generated from solution by an all-glass compressed-air nebulizer. Sizing was performed by determining the titratable acidity.

Airborne mass concentrations were determined by putting two fiberglass filters in series inside the chamber and sampling at constant flow rates for up to 1 h. The first filter captured the aerosol and the second filter gave the change of filter weight due to humidity and allowed the efficiency of the primary sample filter to be verified.

Ozone, produced by passing medical grade oxygen through an electrical ozone generator, was introduced into a chamber run at constant flow rate and slight negative pressure. A Dasibi ultraviolet monitor was used to determine the ozone levels.

All samples for aerosol and gas characterization were acquired from the center of the breathing zone of the animals. Sampling lines were large-bore stainless steel for aerosol with Teflon for ozone.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Remarks on duration:

relative humidity: 39 % (low-humidity exposure); 85 % (high-humidity exposure)

Concentrations:

3.6 mg/m³; MMAD 0.4 µm

No. of animals per sex per dose:

10 - 12 male

Control animals:

yes

Statistics:

two-tailed t-test

Atmospheres

Concentrations and other characteristics of the atmospheres were remarkably stable from one exposure to another. Average data for all runs with standard deviations were: ozone, 0.79 ± 0.02 ppm; aerosol concentrations, 3.6 ± 0.4 mg/m³; low relative humidity, 39 ± 3 %; high humidity, 85 ± 4 %; MMAD of salt aerosols, 0.4 ± 0.1 µm ; and MMAD of sulfuric acid aerosols, 1.0 ± 0.2 µm. The aerosols had average estimated geometric standard deviations of 1.9 -2.3 from impactor data. Electron microscopy indicated geometric standard deviations of 1.6 -1.7.

Clearance Measurements

The low relative humidity sham-exposed animals were selected as primary controls to examine the effect of high relative humidity as a potential cotoxin. A total of 12 groups of 10 - 15 rats each were exposed to low-humidity clean air. The clearance data for these animals are given in Table 1 (see attached file). Some animals were excluded from the data analysis because they did not consume food or water at a sufficient rate to remove tracer particles from the gastrointestinal tract. When this occurred, in about 5% of the rats, clearance half-time values could not be obtained.

The effect of high humidity on clearance was interesting. The short-term clearance half-time was longer in the high-humidity group by 0.9 h ; the long-term clearance half-time was diminished by high relative humidity by about 90 h (significant at p= 0.1).

As shown in Table 2 (see attached file), ozone alone at 0.8 ppm and low humidity statistically significantly slowed early clearance and accelerated late clearance. These effects were even greater at high humidity, the effects of humidity being roughly additive to those of ozone.

Ammonium sulfate at high or low humidity did not have any significant effects on early or late clearance compared to that in low-humidity clean-air controls.

The clearance data for aerosols combined with ozone are very similar to those for ozone alone. In no case is there a statistically significant difference between ozone alone and ozone with an aerosol at the same humidity. Further, in the majority of cases clearance patterns with sulfate particles and ozone both present lie between those for sham-exposed groups and groups exposed to ozone only. The atmosphere with the greatest effect on short-term clearance was sulfuric acid mist with ozone at high humidity.

Interpretation of results:

study cannot be used for classification

Cross-reference 4

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

other information

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

GLP compliance:

not specified

Test type:

other: no data

Species:

dog

Strain:

other: mongrel

Sex:

not specified

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

not specified

Vehicle:

other: no data

Duration of exposure:

7.5 - 240 min

Concentrations:

1.1, 4.1, 9.5 mg/m³

No. of animals per sex per dose:

5 dogs per group

Control animals:

no

Statistics:

Analysis of variance was performed after the model of Lorenzen (1977). In each case the response to an agent was the change in a physiological parameter from its baseline value. Responses to sulfates were compared to responses to NaCl.

First experiment (7.5 -min exposure)

There were no significant alterations in total respiratory resistance, static lung compliance, functional residual capacity, specific total respiratory conductance, and specific lung compliance after breathing for 7.5 min aerosols generated from 0.1 % and 1.0 % solutions of the test substance (mass concentrations: 1.1 and 9.5 mg/m³, respectively).

There were no differences observed when comparing the results from exposure to the test substance with the results of exposure to an aerosol generated from 1.0 % NaCl (mass concentration: 10.0 mg/m³)

Second experiment (4 -hour exposure)

There were no significant alterations in total respiratory resistance, functional residual capacity, static lung compliance, specific lung compliance, and specific total respiratory conductance after 4-h exposures to an aerosol of the test substance. Further, there were no significant alterations in mean pulmonary arterial and carotid arterial pressures, cardiac output, heart rate, stroke volume, arterial pH, and arterial O2 and CO2 tensions.

Interpretation of results:

study cannot be used for classification

Cross-reference 5

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

other information

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

GLP compliance:

not specified

Test type:

other: test on effect of mucociliary clearance

Limit test:

yes

Species:

rabbit

Strain:

other: mixed-breed

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.5 - 2.7 kg
- Housing: in stainless-steel cages
- Diet: standard diet, ad libitum
- Water: ad libitum
- Acclimation period: 1 week
A Plexiglas yoke was placed around the rabbit's neck to prevent head movement during clearance measurements.

ENVIRONMENTAL CONDITIONS
The rabbits were housed in temperature- and humidity-controlled rooms.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: water

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Ammonium sulfate aerosols were generated from dilute (0.03 N) solutions of the chemical using a Laskin nebulizer. The output was mixed with filtered room air which had been temperature and humidity conditioned (24 +/- 0.3 °C, 78 +/- 5 % rel. hum.), and then conveyed into a mixing chamber containing ports for aerosol delivery to the five rabbits. Mass concentration of sulfate was measured in the mixing chamber during each exposure by sampling with Teflon filters, followed by sulfate determination using thermometric titration calorimetry.


TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter): 0.4 µm / GSD (Geometric st. dev.): 1.6

Analytical verification of test atmosphere concentrations:

yes

Remarks:

thermometric titration calorimetry

Duration of exposure:

1 h

Concentrations:

2000 µg/m³ (nominal);
1800, 2200 µg/m³ (actual)
The size (MMD) of the aerosol was 0.4 µm (GSD 1.6).

No. of animals per sex per dose:

5 males

Control animals:

other: A series of 10 air sham-control tests (i.e., exposure for 1-hour to temperature and humidity-conditioned air) were performed on each rabbit prior to any sulfate exposure to obtain baseline values.

Statistics:

t-test

No significant effects of the test substance on mucocilary clearance were observed after exposure to the test substance. For comparison: Ammonium bisulfate exposure produced a significant depression of clearance rate at 1700 mg/m³.

Interpretation of results:

study cannot be used for classification

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium chloride

EC Number:

235-186-4

EC Name:

Ammonium chloride

Cas Number:

12125-02-9

Molecular formula:

ClH4N

IUPAC Name:

ammonium chloride

Results and discussion

Effect levels

Effect levels 1

Sex:

male

Dose descriptor:

LC50

Effect level:

> 3.6 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: rat

Remarks:

(Phalen, 1980)

Effect levels 2

Sex:

not specified

Dose descriptor:

LC50

Effect level:

> 0.81 mg/m³ air

Based on:

test mat.

Exp. duration:

1 h

Remarks on result:

other: guinea pig

Remarks:

(Amdur, 1978)

Effect levels 3

Sex:

not specified

Dose descriptor:

LC50

Effect level:

> 800 mg/m³ air

Based on:

test mat.

Exp. duration:

8 h

Remarks on result:

other: guinea pig

Remarks:

(Pepelko, 1980)

Effect levels 4

Sex:

not specified

Dose descriptor:

LC50

Effect level:

> 9.5 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: dog

Remarks:

(Sackner, 1981)

Effect levels 5

Sex:

male

Dose descriptor:

LC50

Effect level:

> 2.2 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

1 h

Remarks on result:

other: rabbit

Remarks:

(Schlesinger, 1984)