Ammonium chloride

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Toxicity and carcinogenicity of acidogenic or alkalogenic diets in rats; effects of feeding NH4Cl, KHCO3 or KCl

Author:

Lina, B.A.R. and Kuijpers, M.H.M.

Year:

2004

Bibliographic source:

Food and Chemical Toxicology 42, 135 - 153

Materials and methods

Test guideline

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

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

Specific details on test material used for the study:

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 695.7 mg/kg bw/day (nominal)

Remarks:

resembles 2.1 % of the diet

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Results of examinations

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)

Effect levels

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.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

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.