Ammonium chloride

Endpoint summary

Description of key information

In an in vivo repeated dose toxicity study similar to OECD guideline 408 in rats (Lina, 2004), a NOAEL (90 days) of 1695.6 mg/kg bw/d was determined.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

(only two doses tested, only 10 animals/sex/dose, no satellite recovery group, no neurobehavioural tests, no ophthalmological test)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Merck, Darmstadt, Germany (Catalogue No.:1145)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, the Netherlands
- Age at study initiation: 5 weeks of age
- Housing: groups of five of the same sex and the same treatment group, in suspended stainless steel cages with wire-mesh floor and front
- Diet: ad libitum; the Institute’s cereal based rodent diet (Rutten and De Groot, 1992, Food and Chemical Toxicology 30, 601–610).
- Water: ad libitum; tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 35 – 70
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

1 695.7 mg/kg bw/day (nominal)

Remarks:

resembles 2.1 % of the diet

Dose / conc.:

3 372.6 mg/kg bw/day (nominal)

Remarks:

resembles 4 % of the diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical signs and survival were checked daily

BODY WEIGHT: Yes
- Body weights of the individual rats were determined weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food intake of the five rats in each cage was determined weekly over a one week period

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- water intake was recorded daily per cage in weeks 1, 4, 6, 8, 12

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The haematological examinations were conducted in weeks 4, 13
- Anaesthetic used for blood collection: No data
- How many animals: all
- Parameters checked: haemoglobin concentration, packed cell volume, red blood cells, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration, thrombocytes, total white blood cells, prothrombin time and differential white blood cells.

BLOOD GAS ANALYSES
- Time schedule for collection of blood: conducted in weeks 5 and 13
- How many animals: 10/sex/dose
- Parameters checked: comprised determination of pH, pCO2 and pO2 directly after sampling in capillary tubes, and calculation of bicarbonate and base excess.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after at necropsy (after 13 weeks)
- Anaesthetic used for blood collection: yes (ether)
- How many animals: 10/sex/dose
- Parameters checked: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, lactate dehydrogenase, total protein, albumin, total bilirubin, urea, creatinine and calcium, inorganic phosphate, chloride, and sodium and potassium.

GLUCOSE
- Glucose was examined in week 4, and 12 after overnight fasting using whole blood from the tip of the tail.

URINALYSIS: Yes
- Urinary acid indices were determined in weeks 1, 4, 6 and 10 in urine samples obtained from rats placed in metabolism cages with no food and water for 2–3 h at the start of the light period. Urinary pH was measured in individual samples; however mean pH was calculated as the negative logarithm of the mean of hydrogen concentrations instead as the arithmetical mean of the individual pH values. Bicarbonate, titrable acid, ammonia and urea were measured in pooled samples (2/sex/group) and related to creatinine
- Non-fasted (food and water available during sampling), 24 h urine samples were collected from 10 rats/sex/group in week 6. The samples were analysed for volume (graduated tubes) and density, gamma glutamyl transferase, creatinine, urea, calcium, potassium, sodium, phosphate and sulphate. Hydroxyproline excretion was determined in weeks 11 in pooled samples (two samples from five rats each/sex/group) that had been collected in graduated tubes on dry ice during a 24-h period.
- The concentrating ability of the kidneys was examined by collecting urine from 10 rats/sex/group, during the last 16 h of a 24-h water deprivation period in weeks 4, 7 and 13. During the 16-h collection period no feed was available. The samples were analysed for volume (graduated tubes) and density. In addition, examinations for protein, glucose, occult blood, ketones, bilirubin and urobilinogen, appearance and microscopy of the sediment were carried out in the individual samples collected in weeks 4 and 13.

Sacrifice and pathology:

SACRIFICE
At the end of 13-weeks, all rats were killed on a number of successive working days by exsanguination from the abdominal aorta, under light ether anaesthesia.

GROSS PATHOLOGY
- Femur composition was assessed at autopsy at the end of the 13-week in 10 rats/sex/group.
- Any abnormalities were recorded and the adrenals, brain, pituitary, kidneys, liver, spleen, testes, ovaries, thyroid, thymus and heart were weighed. Paired organs were weighed together.

HISTOPATHOLOGY
The following tissues were preserved in 10% neutral phosphate buffered formalin: adrenals, aorta, brain, caecum, colon, epididymides, heart, kidneys, liver, lungs, mesenteric lymph nodes, oesophagus, ovaries, pancreas, parathyroids, pituitary, rectum, small intestine (duodenum, ileum, jejunum), spleen, stomach, testes, thymus, thyroid, urinary bladder, uterus, and all gross lesions.
- Doses examined: all animals of each sex in the control group, the NH4Cl high-dose groups were examined microscopically. Adrenals, heart, kidneys, stomach, mammary glands, thyroid, testes, urinary bladder and uterus were also examined in the low dose group

Statistics:

Numerical data were evaluated for statistical significance using the following statistical tests: one-way analysis of (co)variance [AN(C)OVA] followed by Dunnett’s tests, or least significance difference (LSD) tests. Rates were evaluated by Mann–Whitney’s U test. Histopathological data were evaluated by two-sided Fisher exact probability test. A P value of less than 0.05 was considered to indicate statistical significance.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

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

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

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

Details on results:

CLINICAL SIGNS AND MORTALITY
- There was the usual random incidence of ageing symptoms that occur in this strain of rats when maintained over a period of time, but there were no treatment-related abnormalities in condition or behaviour after 13 weeks.
- Mortality rate was not affected by treatment of NH4Cl. None of the rats died after 13-weeks

BODY WEIGHT AND WEIGHT GAIN
- 4.0 % NH4Cl resulted in significant (p < 0.01) growth depression at the end of 13 weeks (male 17.3 %, female 19.2 % below controls). 2.1 % NH4Cl also markedly decreased growth in females only (11.1 % below control values; p< 0.01).

FOOD INTAKE:
- In the initial phase, food intake was decreased in the 4.0 % dose group. Otherwise, there were no consistent differences in food intake among the groups (data not shown).

WATER INTAKE
- The feeding of 4.0 % NH4Cl resulted in increases in water intake up to 40 – 60 %. With 2.1 % NH4Cl, water intake was ca. 20 % increased in males at all stages but not in females.

BLOOD GAS ANALYSIS
The feeding of NH4Cl was accompanied by a dose related decrease in base excess associated with lower blood pH and bicarbonate concentration. (Data after 13 weeks are not shown in the publication).

URINARY EXAMINATIONS
- 2.1 % NH4Cl caused a consistent decrease in urinary pH values. Urinary pH was reduced by 4.0 % NH4Cl to a similar extent after 13 weeks (data not shown).
- Net acid excretion with NH4Cl was, to a lesser extent, also affected at the 2.1 % NH4 Cl, whereas at the higher level (4.0 %) NH4Cl (13-week) titratable acidity and ammonia excretion were further increased (data not shown in publication).
- The volume of urine collected from rats in the renal concentration test was comparable between the low dose groups and the control group after 6 and 13 weeks in both sexes. Density (g/L) was slightly but significantly reduced (1052 vs 1064 in controls) in males only and only after 6 weeks. After 13 weeks, no differences from controls were seen. Data for high dose animals were not shown in the publication.

HEMATOLOGY and CLINICAL CHEMISTRY
- There were no consistent or treatment-related effects on red blood cell variables, clotting potential or total and differential white blood cell counts in any of the groups.
- Plasma chloride levels were significantly (p < 0.05) increased with 2.1 % NH4Cl after 13 weeks (2.8 % over control) in males only. With 4.0 % NH4Cl the increases were in both sexes ranging from 5.7 - 6.6 % after 13 weeks (p< 0.01)
- Alkaline phosphatase activity was significantly (p < 0.01) increased (both sexes) in comparison to controls with 4.0 % NH4Cl only after 13 week (30.1-41.2%). With 4.0 % NH4Cl, decreases were noted in creatinine concentration and aspartate amino transferase activity, as well as increases in bilirubin and inorganic phosphate concentrations were observed but no data was shown in publication.
- Potassium concentrations were not influenced in any treatment group.

ORGAN WEIGHTS
- There were no consistent or treatment-related changes in the weights of the liver, spleen, ovaries, pituitary, thyroid, thymus or heart. The relative weight of the kidneys (relative to body weight) was increased in rats fed 2.1 % NH4Cl (10 % above control value; p < 0.05; males only were affected) or 4.0 % NH4Cl (12 – 28 %; p < 0.01; both sexes) after 13 weeks. The increase in kidney weights was due to hypertrophy which was not associated with any adverse histopathological findings. On the contrary, there was decreased severity of nephrosis in males fed 2.1 % NH4Cl group in a follow up 2.5 years feeding study (see chapter 7.7), indicating, rather a beneficial effect of NH4Cl administration on the kidneys. The relative weight of the adrenals was increased in males only fed 4.0 % NH4Cl (14.1 % above control value). This is in line with zona glomerulosa hypertrophy seen at this dose.
- The analysis of the femur at the end of the studies did not reveal any effects of NH4Cl on femur weight, calcium content or on total bone substance measured as fat free solid.

GROSSPATHOLOGY
- Macroscopic examination at necropsy did not reveal significant differences among the treatment groups and the controls.

HISTOPATHOLOGY (see attachment)
- Most histopathological changes observed in the various studies were about equally distributed among the treatment groups and the controls and represent normal background pathology for rats of this strain and age. Treatment-related non-neoplastic changes were, however observed. Dose-related increases in the incidence of zona glomerulosa hypertrophy (adrenal) occurred in males and females both sexes
- 2.1 % NH4Cl: Males; 0 vs. 1 case in the controls, females 2 vs. 0 cases in controls (not statistically significant in both sexes)
- 4.0 % NH4Cl: Males; 10 vs. 1 case in the controls (p < 0.01), females: 4 vs. 0 cases in control (not statistically significant in females)

Key result

Dose descriptor:

NOAEL

Remarks:

(90 days; systemic effects)

Effect level:

ca. 1 695.7 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Based on the body weight reduction of up to ca. 20 % seen in both sexes of the high dose group.

Key result

Critical effects observed:

not specified

It may be concluded that most of the observed changes can be regarded as physiological adaptations to the feeding of acid forming salt, and that the rats showed a remarkable adaptive capacity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 695.6 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study comparable to guideline with sufficient documentation for assessment.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a repeated dose toxicity study for up to 13 weeks, ammonium chloride (> 99.5% pure) was administered to 10 Wistar rats/sex/dose in diet at dose levels of 0, 2 % and 4 % ppm (0, 2200 and 4231.1 mg/kg bw/day; based on food consumption) for 4 weeks and 0, 2.1 % and 4 % (0, 1695.6 and 3372.5 mg/kg bw/day; based on food consumption) for 13 weeks. There were no compound related effects on mortality, clinical signs, or gross pathology either after 4 weeks or 13 weeks of feeding. Food consumption was decreased in the high dose group after 4 weeks and in the initial phases in the 13 week animals. Concomitantly, 4 % NH4Cl in the diet resulted in marked and significant (p < 0.01) growth depression at the end of 13 weeks (male 17.3 %, female 19.2 % below controls). 2.1 % NH4Cl also markedly decreased growth in females (11.1 % below control values; p< 0.01). Similar decrease in the body weights of females were seen after 4 weeks of treatment (7.5 % below control value; p < 0.05) with 2.1 % NH4Cl. NH4Cl induced metabolic acidosis in the rats, as shown by decreases in blood pH, base excess and bicarbonate concentration. The renal contribution to the maintenance of the acid-base state of the blood was reflected in decreased urinary pH and increased urinary net acid excretion. Urinary calcium and phosphorus were increased but the calcium and fat free solid content of the femur were not affected. Histopathology examinations revealed dose related increases in the incidence of zona glomerulosa hypertrophy (adrenals) after 13 weeks (2/20 low dose; 14/20 high dose vs. 1/20 controls) which is ascribed to chronic stimulation of the adrenal cortex by NH4Cl induced acidosis. A slight but no significant increase in the incidence of zona glomerulosa was seen after 4 weeks (2/20 low dose; 3/20 high dose vs. 0/20 controls). The relative weight of the kidneys (relative to body weight) was increased in rats of fed 2.1 % NH4Cl (10 % above control value; p < 0.05; male only was affected) or 4 % NH4Cl (12 - 28 %; p < 0.01; both sexes) after 13 weeks but this was not accompanied by a dysfunction of the kidney nor were there any adverse histopathological findings. The NOAEL after 4 weeks is ca. 2200 mg/kg bw/day based on the significant body weight reductions (18 - 25 %) in both sexes of the higher dose animals. The LOAEL (13 weeks) is 3372.5 mg/kg bw/day based on the body weight reduction of up to ca. 20 % seen in both sexes of the high dose group. The NOAEL (13 weeks) is thus 1695.6 mg/kg bw/day (Lina, 2004). This subchronic toxicity study in the rats is acceptable for assessment and is comparable to the OECD test guideline (OECD 408) in rats with restrictions (only two test doses were used and the lack of robust results reporting).

In another study with focus on the bladder toxicity effects of NH4Cl, a group (10 animals/group) of 4 - 5 weeks old, male Sprague Dawley rats were fed a diet containing 0 and 12300 ppm NH4Cl (> 98.7 % pure) (0 and 684 mg/kg bw) for a period of 70 days. After 70 days, the animals were subjected to diets free of the test chemical for up to 7 days before sacrifice. No compound dependent effects on food consumption, body weights, gross and histopathology of the bladder were observed. NH4Cl in the diet caused a reduction of urine pH and an increase in calcium excretion. This is ascribed to direct inhibition of tubular reabsorption of calcium and increased dissociation of plasma protein-bound calcium due to the decrease in plasma pH so that more ionised calcium is filtered through the glomerulus. However, although in humans evidence exists that calcium mobilisation from bone is quantitatively important in metabolic acidosis of less than several weeks of duration, it has been argued on the basis of theoretical predictions, that mobilisation of calcium in large amounts cannot be sustained for very long, because all of the bone in the body would be dissolved within a few years and such losses could obviously not persist. The NOAEL is < 684 mg/kgbw/day (Arnold, 1997). There are a series of other studies performed in rats, rabbits and dogs with focus on bladder toxicity. The main effect seen after subacute or subchronic exposure to NH4Cl was metabolic acidosis.

Justification for classification or non-classification

Repeated Dose Toxicity

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the substance is considered not to be classified for repeated dose toxicity (UN GHS No Category) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

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