Ammonia, anhydrous

Endpoint summary

Description of key information

Ammonia is a gas and testing via the oral route is not feasible. Studies with read-across compounds diammonium phosphate and diammonium sulphate are available and provide useful information on the systemic toxicity of ammonia and its salts. Subacute, subchronic and chronic toxicity testing in rats with these source substances resulted in NOAEL values of 250, 868 and 258 mg/kg bw, which are equivalent to ‘target’ NOAEL values of 68, 225 and 67 mg/kg bw. Dermal effects will be dominated by local corrosion/irritation and significant systemic toxicity is not predicted. The repeated dose toxicity of the substance by inhalation has been adequately tested under a number of non-standard inhalation toxicity studies as weight-of-evidence. The data indicate that the primary effect of exposure to inhaled anhydrous ammonia is local irritation of the respiratory tract.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

There were no mortalities during the study. No clinical signs were observed, including diarrhoea which was noted in the preliminary study. There were no effects on body weight or food consumption, except for a tendency towards increased food intake in the 3.0% males (Table 1).
No group differences were detected in haematology parameters. Some slight changes were found in white blood cell paramter, but there was no dose-relationship and they were therefore considered to be incidental. There were also no effects on serum chemistry parameters.
Absolute and relative kidney weights were increased or showed a tendency for increase at 3.0% in both sexes. Absolute spleen weights were decreased and relative liver weights were increased in the 3.0% male group. No dose-related changes were found in the other organs.
There were no obvious macroscopic findings at necropsy. Several non-neoplastic lesions such as bile duct proliferation in the liver and focal myocarditis in the heart were noted in the control and 3.0% group, and there were no significant differences in their incidences between the groups (in either sex). Malignant pheochromocytoma of the adrenal in the 3.0% males, two adenomas in the anterior pituitary in 3.0% females, and uterine endometrial stromal poly in 1 female control, were noted.

Dose descriptor:

NOAEL

Effect level:

0.6 other: %

Based on:

other: Ammonium sulphate

Sex:

male/female

Basis for effect level:

other: Based on effects on kidney and spleen weights at the 3.0% level. The dietary level of 0.6% is equivalent to 256 and 284 mg/kg/day in males and females, respectively.

Critical effects observed:

not specified

Table 1. Final body weight, food consumption and test substance intake.

Sex	Dose level (%)	Final body weight (g)	Food consumption (g/rat/day)	Intake of ammonium sulphate (mg/kg bw/day)
Male	0	410.9±12.3	13.9	-
	0.1	428.6±17.6	13.6	42
	0.6	416.7±23.7	13.4	256
	3.0	400.5±15.1	15.7	1527
Female	0	207.4±13.5	8.4	-
	0.1	220.3±8.7	8.6	48
	0.6	219.2±13.6	8.4	284
	3.0	212.7±24.4	8.6	1290

Each value represents the mean throughout the administration period.

Conclusions:

Based on effects on organ weights, the NOAEL of Ammonium sulphate was estimated to be 0.6%, which is equivalent to 256 and 284 mg/kg/day in males and females, respectively.

Executive summary:

Chronic toxicity studies (and carcinogenicity; reported in Section 7.7) of read-across substance ammonium sulphate, used as a food additive in fermentation, were performed in male and female Fisher 344 rats at dietary concentrations of 0%, 0.1%, 0.6% and 3.0% in a 52-week toxicity study. Treatment caused significant increase in kidney and/or liver weights in males and females of the 3.0% diet group, but no effects were found on survival rate, body weights, and hematological, serum biochemical or histopathological parameters at any dose levels in the chronic toxicity study. It was concluded that the no observed adverse effect level of ammonium sulphate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg b.w./day in males and females, respectively, and the compound is non-carcinogenic under the conditions of the study.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Male animals in the 3% group exhibited diarrhea during the administration period

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

FOOD CONSUMPTION AND COMPOUND INTAKE
14.2, 14.0, 14.3, 14.1, 13.8 in the males of the 0, 0.38, 0.75, 1.5 and 3% groups, respectively. In females, the values were 9.2, 9.1, 9.3, 9.3 and 8.4 for the 0, 0.38, 0.75, 1.5 and 3% groups, respectively.

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

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

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

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

Dose descriptor:

NOAEL

Effect level:

1.5 other: % in diet [Equivalent to 886 mg/ kg bw/day]

Based on:

other: Ammonium sulphate

Sex:

male

Basis for effect level:

other: Male animals in the 3% group exhibited diarrhoea during the administration period.

Dose descriptor:

NOAEL

Effect level:

3 other: % in diet [equivalent to 1975 mg/ kg bw/d]

Based on:

other: Ammonium sulphate

Sex:

female

Basis for effect level:

other: no effects

Critical effects observed:

not specified

Ammonium sulphate was shown to be of low toxicity. Based on these results, the NOEL was judged to be 1.5% in males (886 mg/kg bw/day) and 3% in females (1975 mg/kg bw/day).

Conclusions:

Ammonium sulphate was shown to be of low toxicity. Based on these results, the NOEL was judged to be 1.5% in males (886 mg/kg bw/day) and 3% in females (1975 mg/kg bw/day).

Executive summary:

A 13-week subchronic oral toxicity study with read-across substance ammonium sulphate was performed in both sexes of F344 rats by feeding them diet containing concentrations of 0%, 0.38%, 0.75%, 1.5%, and 3.0% of the substance. Rats were randomly divided into 5 groups each consisting of 10 males and 10 females. Male animals in the 3% group exhibited diarrhoea during the administration period. No changes indicating obvious toxicity were observed in the body weights, organ weights, hematological, serum biochemical, or histopathological examinations. Based on these results, the NOEL (no-observed-effect level) of ammonium sulphate in F344 rats was judged to be 1.5% in males (equivalent to 886 mg/kg bw/d) and 3% in females (1975 mg/kg bw/d). The MTD for 2-year carcinogenicity studies in F344 rats was concluded to be 3.0% or more in the diet.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

Toxicity subgroup:
There were no treatment-related deaths and no signs of overt clinical toxicity. Bodyweight gain and food consumption appeared to be suppressed among males at 1,500 mg/kg/day (bw 78% of control). The only treatment-related change evident in haematology was a reduction in activated partial thromboplastin time for males at 750 or 1,500 mg/kg/day (74 and 76% of control, resp.). There was a slightly greater variability in blood chemistry parameters, and the following were blood chemistry changes for males that may represent an effect of treatment: a non dosage-dependant elevation of alkaline phosphatase levels at 750 and 1,500 mg/kg/day (132 and 131% of control, resp.); reduced glucose and phosphorous levels at 1,500 mg/kg/day (79 and 82% of control, resp.); a dosage-dependant reduction in total protein at 750 and 1,500 mg/kg/day (93 and 91% of control, resp.) with a slightly elevated albumin/globulin ratio at the high dose (117% of control). Changes in females were limited to a decrease in phosphorous levels and a non-significant increase in alkaline phosphatase levels at 1,500 mg/kg/day (81 and 122% of control, resp.). No effect was observed on functional observations. Reddening of the extremities during the first week of dosing was observed in all doses, including 250 mg/kg/day but reduced as the treatment period progressed. Histological examination of the stomachs revealed some submucosal inflammation at all doses (0/5, 3/5, 4/5 and 2/5 for males and 0/5, 2/5, 4/5 and 4/5 for females at 0, 250, 750 and 1500 mg/kg bw), but this change was not dose depend
ent and was not statistically significant at the low dose.

Reproductive subgroup:
There were no treatment-related deaths and no signs of overt clinical toxicity. Bodyweight gain for reproductive subgroup females receiving 1,500 mg/kg/day was reduced during the first week of gestation (82% of control), after which they returned to levels comparable to the controls for the remainder of the study. Mating performance and fertility were unaffected by treatment, and parental treatment had no apparent effect on the offspring to day 4 of age.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

other: Diammonium phosphate

Sex:

male/female

Basis for effect level:

other: general toxicity

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day (nominal)

Based on:

other: Diammonium phosphate

Sex:

male/female

Basis for effect level:

other: Reproduction/developmental toxicity

Dose descriptor:

LOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

other: Diammonium phosphate

Sex:

male/female

Basis for effect level:

other: General toxicity

Dose descriptor:

LOAEL

Effect level:

> 1 500 mg/kg bw/day (nominal)

Based on:

other: Diammonium phosphate

Sex:

male/female

Basis for effect level:

other: Reproduction/developmental toxicity

Critical effects observed:

not specified

NOAEL: 250 mg/kg/day (general toxicity); 1,500 mg/kg/day (reproduction/developmental toxicity)

LOAEL: 750 mg/kg/day (general toxicity); >1,500 mg/kg/day (reproduction/developmental toxicity)

Conclusions:

Diammonium phosphate NOAEL: 250 mg/kg/day (general toxicity); 1,500 mg/kg/day (reproduction/developmental toxicity)
Diammonium phosphate LOAEL: 750 mg/kg/day (general toxicity); >1,500 mg/kg/day (reproduction/developmental toxicity)

Executive summary:

The toxicity of read-across substance diammonium phosphate (DAP) was assessed in a combined repeated dose toxicity study with a reproduction / developmental toxicity screening test in rats. Clinical biochemistry changes were observed in males and females (males: elevation of alkaline phosphatase levels at 750 and 1,500 mg/kg/day; reduced glucose and phosphorous levels and total protein at 750 and 1,500 mg/kg/day with a slightly elevated albumin/globulin ratio at the high dose; females: decrease in phosphorous levels and increase in alkaline phosphatase levels at 1,500 mg/kg/day)The NOAEL was found to be: 250 mg/kg/day (general toxicity); 1,500 mg/kg/day (reproduction/developmental toxicity). The LOAEL was found to be: 750 mg/kg/day (general toxicity); >1,500 mg/kg/day (reproduction/developmental toxicity)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

68 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Studies with read-across compounds are available and provide useful information on the systemic toxicity of ammonia and its salts.

System:

hepatobiliary

Organ:

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Older published study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Subchronic inhalation toxicity of ammonia in the rat

GLP compliance:

no

Remarks:

: older published study, pre-dates GLP

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Male Wistar rats, aged 40-45 days old and weighing 90-140 g at study initiation. The animals were housed singly or in groups of up to 8 in plastic cages with metal lids. Formulation R feed pellets (VEB Versuchstierproduktion, Berlin) and water were provided ad libitum. Rats were randomly assigned to treatment groups.

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not reported

Details on inhalation exposure:

Rats were exposed to ammonia continuously for 50 days, in an climatic chamber with a volume of 250 L (maximum of 15 animals per chamber). Ammonia was fed into the chamber using a control valve. The temperature of the chamber was approx. 22°C, and relative humidity 50-70%.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A gas analysis applicance was used to measure the ammonia concentration; the measured value was taken to an instantaneous adjustor which controlled the valve according to the desired theoretical value.

Duration of treatment / exposure:

50 days

Frequency of treatment:

Continuous throughout exposure period

Dose / conc.:

35 mg/m³ air

Dose / conc.:

63 mg/m³ air

No. of animals per sex per dose:

8-14 males per group

Control animals:

yes, concurrent no treatment

Details on study design:

Male Wistar rats were exposed to 35 or 63 mg/m3 ammonia continuously for 50 days.

Positive control:

Not examined

Observations and examinations performed and frequency:

Clinical signs, body weight gain, food intake, haemaglobin, haematocrit, erythrocyte count, total and differential leucocyte count.

Sacrifice and pathology:

Total protein, lung and liver weights.

Other examinations:

No other examinations reported.

Statistics:

Bartlett's test and Scheffe's test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

There were no treatment related clinical effects, and no mortality occurred. Body weight gain was 105% and 95% of the control for the 35 and 63 mg/m3 groups, respectively. Food intake was 94% and 108% of the control for the 35 and 63 mg/m3 groups, respectively. Increased haemaglobin and haematocrit were seen at 63 mg/m3 compared to controls. There were no treatment related effects on organ weights.

Dose descriptor:

NOAEC

Remarks:

50 days

Effect level:

35 mg/m³ air

Sex:

male

Basis for effect level:

other: haematology parameters

Critical effects observed:

not specified

No further information       

Conclusions:

The NOAEC was 35 mg/m3 ammonia in air.

Executive summary:

Male Wistar rats were exposed to two concentrations of ammonia gas, continuously for 50 days. Concurrent controls remained untreated. There was no mortality at either concentration (35 or 63 mg/m³), and no treatment-related clinical effects were observed. Body weight gain and food intake, as compared to control values, was not significantly affected by ammonia exposure. At 63 mg/m³ rats showed increased haemoglobin and haematocrit levels compared to controls. The NOAEC was 35 mg/m³.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Older published study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Subchronic exposure to ammonia gas

GLP compliance:

no

Remarks:

: older, published study - pre-dates GLP

Species:

guinea pig

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

Twelve male guinea pigs, plus 6 controls. Animals were fed Purina rabbit chow supplemented with greens. Food and drink were withheld during exposure periods.

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not reported

Details on inhalation exposure:

Guinea pigs were exposed to anhydrous ammonia gas released into the air of an exposure chamber. Controls were not subjected to a mock-exposure regimen.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of ammonia in the chamber was measured 2 or 3 times daily by chemical analysis. Actual concentrations were reported to be 140 - 200 ppm.

Duration of treatment / exposure:

18 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
98 - 140 mg/m3 (average concentration 119 mg/m3)
Basis:
no data

No. of animals per sex per dose:

Twelve male guinea pigs plus 6 controls.

Control animals:

yes

Details on study design:

Guinea pigs were exposed to an average concentration of 119 mg/m³ ammonia in air for 18 weeks (6 hours/day, 5 days/week). At 6 week intervals 4 experimental and 2 controls animals were sacrificed and subject to necropsy.

Positive control:

A positive control was not examined.

Observations and examinations performed and frequency:

Body weights were determined weekly.

Sacrifice and pathology:

Necropsy was performed at 6, 12 and 18 weeks. The heart, lungs, liver, stomach, small intestine, spleen, kidneys and suprarenal glands were examined microscopically.

Other examinations:

No other examinations reported.

Statistics:

Statistical analysis was no performed

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

There were no significant findings at necropsy in the animals sacrificed at 6 and 12 weeks. In animals sacrificed at 18 weeks there was mild congestion of the liver, spleen and kidneys with degenerative changes in the adrenal glands, and hemosiderosis in the spleen indicating hematotoxicity. There was cloudy swelling in the epithelium of the proximal tubules of the kidney as well as albumin precipitation in the lumen with some casts.

Dose descriptor:

LOEL

Remarks:

18 weeks

Effect level:

119 mg/m³ air

Sex:

male

Basis for effect level:

other: Abnormal findings at gross necropsy

Critical effects observed:

not specified

No further information

Conclusions:

18 weeks exposure to ammonia gas resulted in abnormal findings at gross necropsy. No abnormal changes occurred after 6 and 12 weeks exposure. The average exposure concentration was 119 mg/m3.

Executive summary:

Twelve male guinea pigs (plus 6 controls) were exposed to anhydrous ammonia gas for up to 18 weeks (6 hours per day, 5 days per week). The average concentration in air was 119 mg/m³. Four experimental and 2 control animals were sacrificed at 6 week intervals throughout the study. There were no significant findings at necropsy after 6 and 12 weeks exposure. In animals sacrificed after 18 weeks, there was mild congestion of the liver spleen and kidneys, with degenerative changes in the adrenal glands, and hemosiderosis in the spleen indicating hematotoxicity. There was cloudy swelling in the epithelium of the proximale tubules of the kidney as well as albumin precipitation in the lumen with some casts.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Older published study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Exposure of rats to environments containing ammonia for 4-6 weeks

GLP compliance:

no

Remarks:

: older, published study - pre-dates GLP

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

not specified

Details on test animals or test system and environmental conditions:

Young, adult pathogen-free rats of Sherman and Fischer (F344) substrains. They were aged 2-4 months old, and test groups were matched according to age, sex and number (5 or 6 rats) per cage. Rats were fed autoclaved diet for axenic rodents (Allied Mills or Purina) and given sterile tap water ad libitum.

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not reported

Details on inhalation exposure:

The rats were exposed to ammonia from natural sources (soiled bedding in shoebox cages) at an average concentration of 150 ppm (105 mg/m3) for 75 days, and to purified ammonia at 250 ppm (175 mg/m3) for 35 days. The rats were exposed continuously in their housing environment. The temperature was 23.6±0.5°C, and relative humidity was 69±5.0%.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Ammonia levels were monitored daily using NH3 detectors (Honeywell or Kitagawa).

Duration of treatment / exposure:

35 or 75 days

Frequency of treatment:

Continuous throughout treatment period.

Dose / conc.:

105 mg/m³ air

Dose / conc.:

175 mg/m³ air

No. of animals per sex per dose:

12 rats/sex/group

Control animals:

yes

Details on study design:

Rats were inoculated intranasally with colony-forming untis of M. pulmonis. They were housed in environments containing ammonia maintained at specific concentrations from 25 to 250 ppm (only average concentrations are reported). Rats that had not been infected with M. pulmonis were also exposed to ammonia.

Positive control:

Not examined

Observations and examinations performed and frequency:

Clinical signs were reported.

Sacrifice and pathology:

Histological examination of the nasal cavity was carried out.

Other examinations:

No other examinations reported.

Statistics:

Wilcoxon's two-sample rank test, chi-square, and regression and correlation analysis.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

Ammonia exposure (from both natural and purified sources) significantly increased the severity of the rhinitis, otitis media, tracheitis and pneumonia (including bronchiectasis) characteristic of murine respiratory mycoplasmosis (rats infected with M. pulmonis). The prevalence of pneumonia showed a strong tendency to increase directly with environmental ammonia concentration.
Rats not infected with M. pulmonis, developed anatomic lesions limited to the nasal passaged following ammonia exposure.
Histological changes in the olfactory and respiratory epithelia of the nasal cavity were similar for all exposed rats, showing increased thickness, pyknotic nuclei, and hyperplasia. The submucosa were oedematous with marked dilation of the small vessels. Lesions decreased posteriorly.

Dose descriptor:

LOAEL

Remarks:

75 days

Effect level:

105 mg/m³ air

Sex:

not specified

Basis for effect level:

other: histological changes in the nasal cavity

Dose descriptor:

LOAEL

Remarks:

35 days

Effect level:

175 mg/m³ air

Sex:

not specified

Basis for effect level:

other: histological changes in the nasal cavity

Critical effects observed:

not specified

No further information

Conclusions:

The LOEL was an average exposure level of 105 mg/m3 over 75 days.

Executive summary:

Sherman and Fischer rats were exposed to environmental ammonia, derived from natural sources for 75 days, or to purified ammonia for 35 days. Rats were either inoculated intranasally with M. pulmonis prior to exposure, or left untreated. The average ammonia concentrations were 105 mg/m³ for 75 days and 175 mg/m³ for 35 days exposure.

Ammonia exposure (from either source) significantly increased the severity of the rhinitis, otitis media, tracheitis and pneumonia (including bronchiectasis) characteristic of murine respiratory mycoplasmosis (rats infected with M. pulmonis). The prevalence of pneumonia showed a strong tendency to increase directly with environmental ammonia concentration.

Rats not infected with M. pulmonis, developed anatomic lesions limited to the nasal passaged following ammonia exposure.

Histological changes in the olfactory and respiratory epithelia of the nasal cavity were similar for all exposed rats. The LOEL was an average exposure level of 105 mg/m³ for 75 days.

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Continuous inhalation exposure to ammonia gas; toxicity study in the rat

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

Young male specific-pathogen-free rats. Test and control groups were age and weight matched.

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not relevant (gas)

Details on inhalation exposure:

Rats were continuously exposed to atmospheric ammonia gas at a specific concentration for up to 8 weeks.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Up to 8 weeks

Frequency of treatment:

Continuous throughout treatment period

Remarks:

Doses / Concentrations:
350 mg/m3
Basis:
no data

No. of animals per sex per dose:

27 male rats per group

Control animals:

yes

Details on study design:

The rats were sacrificed after different times of exposure (up to 8 weeks) to establish a time dependency of the effects. The continuous atmospheric concentration of 350 mg/m3 was chosen, after the authors noted that general toxic effects (particularly on growth rate) were not found at 175-210 mg/m3.

Positive control:

Not examined

Observations and examinations performed and frequency:

Weight, growth and food intake were measured throughout the exposure period.

Sacrifice and pathology:

Rats were sacrificed at various intervals throughout the exposure period and subject to necropsy.

Other examinations:

No information available

Statistics:

No information available

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Nasal irritation appeared after 4 days exposure. After 3 weeks, the exposed rats showed nasal irritation and inflammation of the upper respiratory tract but no effects were observed in the bronchioles and alveoli. The number of pulmonary alveolar macrophages was similar to that in the controls. After 8 weeks exposure, none of these inflammatory lesions were present.
Weight, growth and food intake were reduced in exposed rats throughout the exposure period. Lungs and kidney weights were significantly increased in exposed rats, liver weight was not affected.

Dose descriptor:

NOAEC

Effect level:

350 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: adverse signs appeared after 4 days of continuous exposure

Critical effects observed:

not specified

No further information

Conclusions:

Continuous exposure to atmospheric ammonia at 350 mg/m3 resulted in irritation and inflammatory effects after 3 weeks, effects were not present after 8 weeks exposure.

Executive summary:

Twenty seven male rats, along with 27 age and weight matched controls, were exposed to atmospheric ammonia gas at a concentration of 350 mg/m³ for up to 8 weeks. The rats were sacrificed after different exposure times. Nasal irritation began on the fourth day. After 3 weeks continuous exposure exposed rats showed nasal irritation and inflammation of the upper respiratory tract. The number of pulmonay alveolar macrophages was similar to that in the controls. After 8 weeks none of the inflammatory lesions were present. Weight, growth and food intake were reduced in exposed rats throughout the exposure period. Lungs and kidney weights were significantly increased in exposed rats, liver weight was not affected.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

35 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

A number of non-standard studies of varying duration, conducted in different species are available.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

35 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

A number of non-standard studies of varying duration conducted in different species are available.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose oral toxicity

Since ammonia is a gas, repeated oral dose testing is not feasible and oral exposure is not relevant. However studies using read-across compounds diammonium phosphate and diammonium sulphate are available and have been evaluated since they provide useful information on the systemic toxicity of ammonia and its salts.

 

In a key four -week combined repeated dose toxicity and reproduction/developmental toxicity screening test (OECD Test Guideline 422) conducted in rats using diammonium phosphate, only minor effects on weight gain and clinical chemistry parameters (males: elevation of alkaline phosphatase levels at 750 and 1,500 mg/kg/day; reduced glucose and phosphorous levels and total protein at 750 and 1,500 mg/kg/day with a slightly elevated albumin/globulin ratio at the high dose; females: decrease in phosphorous levels and increase in alkaline phosphatase levels at 1,500 mg/kg/day) were observed (HLS, 2002). In the study, a NOAEL of 250 mg/kg bw/day was determined for general toxicity, equivalent to a ‘target’ NOAEL of 0.258 x 250 mg/kg bw = 68 mg/kg bw.

 

The subchronic repeated oral dose toxicity of diammonium sulphate has been investigated in rats in a 90 -day study conducted according to OECD Test Guideline 408 (Tagaki et al., 1999) In the study, rats were dosed with diammonium sulphate via the diet at concentrations of 0%, 0.38%, 0.75%, 1.5% and 3.0%. Only minor effects were observed at high dose levels (diarrhoea, renal pathology); a NOAEL of 886 mg/kg bw/day was determined, equivalent to a ‘target’ NOAEL of 0.258 x 868 mg/kg bw = 225 mg/kg bw/day.

 

Chronic toxicity (52-week combined with carcinogenicity) studies with diammonium sulphate in Fisher 344 rats at dietary concentrations of 0%, 0.1%, 0.6% and 3.0% caused a tendency towards increased food intake in the 3.0% males and significant increase in kidney and/or liver weights in males and females of the 3.0% diet group, but no effects were found on survival rate, body weights, and hematological, serum biochemical or histopathological parameters at any dose levels in the chronic toxicity study. It was concluded that the NOAEL of diammonium sulphate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg b./day in males and females, respectively. The NOAEL of 256 mg/kg bw/day (was equivalent to ‘target’ NOAEL of 0.258 x 256 = 67 mg/kg bw/day) was determined in both a one year study (OECD Test Guideline 452) and in two year studies (OECD Test Guideline 453; Ota et al., 2006).

 

Repeated dose dermal toxicity

No data are available. The substance is classified as corrosive: dermal effects will be dominated by local (site of contact) irritation and corrosion: significant systemic toxicity is not expected.

 

Repeated exposure inhalation toxicity

A number of non-standard studies of varying duration which have been conducted in different species are available. The data indicate that the primary effect of exposure to inhaled anhydrous ammonia is local irritation of the respiratory tract.

 

In a supporting 5-week study in pigs, ammonia concentrations had a highly significant adverse effect upon feed consumption and average daily weight gain (Stombaugh et al., 1969). However, there was no significant effect upon the efficiency of food conversion. During both trials the high ammonia levels appeared to cause excessive nasal, lacrimal and mouth secretions. This was more pronounced at 100 and 150 ppm than at 50 ppm. Autopsies carried out on three animals showed no significant gross or microscopic differences related to ammonia level. Cultures of Corynebacterium and Pasteurella were obtained from swabs of the ethmoid turbinates from two animals removed from the compartment maintained at 150 ppm and one animal maintained at 100 ppm. There was no evidence of these bacteria in turbinate swabs from other animals (Stombaugh et al., 1969). NOAEL was determined at 61 ppm.

 

Following studies were used in a weight-of-evidence approach. Various concentrations have been tested in different species (rats, guinea-pigs).

 

In a study, Sherman and Fischer rats were exposed to environmental ammonia, derived from natural sources for 75 days, or to purified ammonia for 35 days (Broderson et al.,1976). Rats were either inoculated intranasally with M. pulmonis prior to exposure, or left untreated. The average ammonia concentrations were 105 mg/m³ for 75 days and 175 mg/m³ for 35 days exposure. Ammonia exposure (from either source) significantly increased the severity of the rhinitis, otitis media, tracheitis and pneumonia (including bronchiectasis) characteristic of murine respiratory mycoplasmosis (rats infected with M. pulmonis). The prevalence of pneumonia showed a strong tendency to increase directly with environmental ammonia concentration .

 

In an 8 week study, twenty seven male rats, along with 27 age and weight matched controls, were exposed to atmospheric ammonia gas at a concentration of 350 mg/m³ (Richard et al,1978). The rats were sacrificed after different exposure times. Nasal irritation began on the fourth day. After 3 weeks of continuous exposure exposed rats showed nasal irritation and inflammation of the upper respiratory tract. The number of pulmonary alveolar macrophages was similar to that in the controls. After 8 weeks none of the inflammatory lesions were present.

 

In an 18 week study twelve male guinea pigs (plus 6 controls) were exposed to anhydrous ammonia gas (6 hours per day, 5 days per week; Weatherby, 1952). The average concentration in air was 119 mg/m³. Four experimental and 2 control animals were sacrificed at 6 week intervals throughout the study. There were no significant findings at necropsy after 6 and 12 weeks exposure. In animals sacrificed after 18 weeks, there was mild congestion of the liver spleen and kidneys, with degenerative changes in the adrenal glands, and hemosiderosis in the spleen indicating hematotoxicity. There was cloudy swelling in the epithelium of the proximal tubules of the kidney as well as albumin precipitation in the lumen with some casts.

 

In a 50-day study male Wistar rats were exposed to two concentrations of ammonia gas, continuously for 50 days (Stolpe & Sedlag, 1976). Concurrent controls remained untreated. There was no mortality at either concentration (35 or 63 mg/m³), and no treatment-related clinical effects were observed. Body weight gain and food intake, as compared to control values, was not significantly affected by ammonia exposure. At 63 mg/m³ rats showed increased haemoglobin and haematocrit levels compared to controls. The NOAEC was 35 mg/m³.

Justification for classification or non-classification

Ammonia is not classified for repeated dose toxicity according to the harmonised classification in Part 3 of Annex VI of the CLP Regulation 1272/2008/EC. The available data do not indicate that the classification of the substance for repeated dose or target organ toxicity according to Regulation 1272/2008/EC is warranted; there is no evidence of marked repeated dose systemic toxicity following exposure to ammonia or ammonium salts.