Ammonium chloride

Carcinogenicity

Description of key information

According to the result of a carcinogenicity study according to OECD Guideline 451, ammonium chloride is negative for carcinogenicity. No carcinogenic effect at the highest dose of 1104.6 mg/kg bw was observed (Lina et al. 2004).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

(only two doses tested, no satellite recovery group, no neurobehavioural tests, no opthalmological tests)

GLP compliance:

not specified

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
- Weight at study initiation: no data
- Fasting period before study: no data
- 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 (%): 30 - 70
- Air changes (per hr): at least 10
- 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:

males: 122 weeks
females: 131 weeks

Frequency of treatment:

daily (continuously)

Post exposure period:

no

Dose / conc.:

502.8 mg/kg bw/day (actual dose received)

Remarks:

resembles to 1.0 % in diet, calculated from food consumption

Dose / conc.:

1 104.6 mg/kg bw/day (actual dose received)

Remarks:

resembles to 2.1 % in diet, calculated from food consumption

No. of animals per sex per dose:

- 18 months treatment: 10
- 30 months treatment: 50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results of preceeding 4 and 13 week feeding studies. Because 4% NH4Cl resulted in marked (ca. 20 %) growth depression, lower dietary levels of NH4Cl were applied in the chronic studies.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical signs and survival were checked daily. In addition, grossly visible or palpable masses were recorded at regular intervals.

BODY WEIGHT: Yes
- Body weights of the individual rats were determined weekly or once every 4 weeks (from 3 months)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food intake was determined per cage (five rats/cage) weekly or once every 4 weeks over 1-week periods (from 3 months)

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- In the various studies, water intake was recorded daily per cage in weeks 1, 4, 6, 8, 12, 35, 55 and 75.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The haematological examinations were conducted in weeks 4, 13, 32, 55 and 75
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10/sex/dose
- 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, 14, 32, 56, 76 and 121 in blood collected from the tip of the tail
- How many animals: 10/sex/dose
- Parameters checked: pH, pCO2 and pO2, calculation of bicarbonate and base excess.
The absolute change in blood bicarbonate does not indicate the exact magnitude of non-respiratory acid-base disorder because of the presence of other buffers in the blood. Therefore, base excess was calculated to assess this magnitude. Base excess is defined theoretically as the amount of strong acid required under standardised conditions to titrate 1 L oxygenated blood to pH 7.4. In practice, it is derived from bicarbonate, pH, and haemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4-weeks, 13-weeks 17 weeks, 38 weeks and 79 weeks
- Animals fasted: no data
- How many animals: 10/sex/dose
- Method: 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.

URINALYSIS: Yes
Parameters: Urinary acid indices were determined in weeks 1, 4, 6, 10, 14, 36, 57, 76 and 129 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. Mean pH values were calculated (- log H+). Bicarbonate, titratable acid, ammonia and urea were measured in pooled samples (two samples/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 weeks 6, 16, 38 and 78. The samples were analysed for volume (graduated tubes) and density, gamma glutamyl transferase, creatinine, urea, calcium (atomic absorption), potassium, sodium, phosphate, and sulphate.Hydroxyproline excretion was determined in weeks 11 and 76, in pooled samples (two samples from five rats each/sex/group).
- 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, 13, 14, 36, 57, 77 and 129. During the 16-h collection period no feed was available. The samples were analysed for volume and densitt. 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, 13, 77 and 129.
- In week 53, six rats/sex/group were placed in metabolism cages. Faeces and urine were collected over a 4-day period, and the excretion of calcium and phosphorus (atomic absorption) was determined and related to the dietary intake.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE
After 18 months, 122 weeks (males) and 131 weeks (females), all surviving animals were killed by exsanguination from the abdominal aorta, under light ether anaesthesia in the respective studies.

GROSS PATHOLOGY
- 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 (not examined at 30 month sacrifice), axillary lymph node (not examined at 30 month sacrifice),brain, caecum, coagulating glands, colon, epididymides, exorbital lachrimal glands, heart, knee joint (not examined at 18 month sacrifice) eye (not examined at 30 month sacrifice), kidneys, liver, lungs, mammary glands, mesenteric lymph nodes, oesophagus, ovaries, pancreas, parathyroids, parotid salivary glands, pituitary, preputial/clitoral glands, prostate, rectum, seminal vesicles, skeletal muscle (not examined at 30 sacrifice), skin (not examined at 30 month sacrifice), small intestine (duodenum, ileum, jejunum), spleen, sternum with bone marrow (not examined at 30 month sacrifice), stomach, submaxillary salivary glands (not examined at 18 month sacrifice), sublingual salivary glands (not examined at 18 month sacrifice), testes, thymus, thyroid, urinary bladder, uterus, Zymbals glands (not examined at 18 month sacrifice) and all gross lesions. The lungs and urinary bladder were inflated with the fixative.
- Doses examined: Paraffin-embedded, 5 mm sections of the above tissues from all animals of each sex in the control group, and the NH4Cl high-dose group were stained with haematoxylin and eosin and examined microscopically. Adrenals, heart, kidneys, stomach, mammary glands, thyroid, testes, urinary bladder and uterus were also examined in intermediate-dose groups.

Other examinations:

Femur composition was assessed at autopsy after 30 months in all surviving rats. The right femur was removed, cleaned from adherent soft tissue and weighed. After defatting, the calcium content was determined by atomic absorption spectrophotometry.

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. p < 0.05 was considered to indicate statistical significance.

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 in the study. Also the type and incidence of palpable masses noted during the chronic studies did not indicate any treatment-related effects.
- In the chronic feeding durations, mortality rate was not affected by treatment with NH4Cl. The overall mortality rate after 30 months was 62, 40 and 52 for males, and 69, 68 and 64 and for females of the control, low and high-dose NH4Cl groups, respectively.

BODY WEIGHT AND WEIGHT GAIN
- Body weights were also statistically significantly decreased in females of the 1.0 % NH4Cl group at various stages of the 30-month study (data not shown). However terminal weights of surviving females, though less than for controls was no longer reduced with significance. After 30 months, mean body weights were minimally decreased in surviving males (by 2.5 %) and females (by 5.5 %) of the high dose group. The decreases were not statistically significant.

FOOD CONSUMPTION:
There were no consistent differences in food intake among the groups (data not shown).

WATER CONSUMPTION:
With 2.1 % NH4Cl, water intake was ca. 20 % increased in males at all stages but not in females. Water consumption was not noticeably affected by 1.0 % NH4Cl (data not shown).

HAEMATOLOGY
- 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 (data not shown)

CLINICAL CHEMISTRY
- Plasma chloride levels were very slightly increased in both sexes with 2.1 % NH4Cl, in week 17 (1.9 - 3.8 % over control values; p< 0.05) and week 38 (2.9 - 3.8% over controls values; p< 0.05) and in week 79 (4.7 % over controls values; p< 0.01) in females fed 1.0 % NH4Cl. Alkaline phosphatase activity was increased (by 20 % compared to controls; p< 0.05) in males only fed 2.1 % NH4Cl in week 17 only. Creatinine concentration and aspartate amino transferase activity, as well as bilirubin and inorganic phosphate concentrations were not affected with 2.1 % NH4Cl in the chronic studies (data not shown).

FAECAL, URINARY AND FEMORAL CALCIUM EXAMINATIONS
- During the 4-day collection period in week 53, it was shown, that 2.1 % NH4Cl significantly raised the urinary output of calcium from 1 to 2 % and 4 to 7 % of the total calcium intake, and the urinary output of phosphorus from 25 to 31 % of the phosphorus intake. Faecal calcium and phosphorus excretion were lower in females, but the changes were not statistically significant and no clear conclusions could be drawn from these data (data not shown).
- Calcium and phosphate excretion showed dose-related increases in rats fed 1.0 and 2.1% NH4Cl and urinary urea excretion was increased with NH4Cl. Acid stress induced hypercalciurea 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. Also, increased mobilisation of calcium from bone salts in order to buffer the pH assault has been postulated in the literature. 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. In this study, the analysis of the femur after 30 months did not reveal any effects of NH4Cl on femur weight, calcium content or on total bone substance measured as fat free solid in rats (see attachment 2), hence the findings in this study do not support the hypothesis that bone mineral participates in chronic acidosis.

URINANALYSIS
- Urinary pH was consistently and significantly (p < 0.01) decreased in rats fed 1.0 and 2.1 % NH4Cl ranging from pH 5.5 - 5.7 % after 1 week to pH 5.5 - 5.6 after 129 weeks. pH range of the control group ranged from 7.7 in week 1 to 6.1 in week 129. The pH was similarly influenced by both doses tested in the 30 month study. The net acid excretion by the kidneys can be assessed as: Net acid excretion = NH4+ excretion + urinary titratable acidity bicarbonate excretion. In both sexes, the 2.1 % NH4Cl diet induced a consistently increased (p < 0.01) titratable acidity (titratable acidity/creatinine; mol/mol) ranging from 2.3 - 5.5 at week 6 (control range 0.0 - 0.6) to 7.8 - 8.1 at week 129 (control range 3.1 -4.0) and ammonia excretion (p < 0.01) (NH4/Creatinine; mol/mol) ranging from 38.4 - 43.9 at week 6 (control range 5.9 - 6.2) to 16.9 - 44.2 at week 129 (control range 5.8 - 8.6). Net acid excretion with NH4Cl was - to a lesser extent - also affected at the respective low-dose levels (see attachment 1). Exogenous ammonium can be metabolised via the urea cycle. The increase in urinary urea excretion observed with NH4Cl is presumed due to the increased NH4+ load which is partly converted to urea and partly excreted as urinary NH4 ions. Urinary bicarbonate excretion was not influenced by NH4Cl feeding (see attachment 1)
The volume of the urine collected during 24 h (food and water available), was generally increased in rats fed 2.1 % NH4Cl. In females, this finding was no longer apparent in the later stages of the study, which may have been due to the high values obtained in the control group. The urinary density did not show consistent differences but tended to be decreased in males of these groups (data not shown).
There were no consistent differences in 24 h excretion of sulphate, gamma glutamyl transferase or hydroxyproline among the groups. Brownish discoloration of the urine and haematuria, as detected with urinary test strips and by microscopic examination of the urinary sediment were occasionally increased in rats fed NH4Cl, but there were no consistent or dose-related differences in incidence or severity of haematuria among the groups. From 18 months (week 78 and 129), the incidence of granular casts in the urinary sediment was increased with 2.1 % NH4Cl in both sexes. The occurrence of crystals was not affected in any group.

GROSSPATHOLOGY
- Macroscopic examination at necropsy after 18 and 30 months did not reveal significant differences among the treatment groups and the controls.

ORGAN WEIGHTS (see attachment 3)
The relative weight of the kidneys (relative to body weight) was increased and remained relatively high with 2.1 % NH4Cl at the subsequent stages (see attachment 3). The increase in renal size is reflective of an increased hypertrophy without increased cell number. Hypertrophy was not associated with adverse histopathological changes. Hypertrophy is a consequence of the NH4Cl acidosis induced renal ammoniagenesis. At several stages, the relative weights of brain and testes were increased in rats showing growth retardation, but these findings were ascribed to the lower body weights of these rats. There were no consistent or treatment-related changes in the weights of the liver, spleen, ovaries, pituitary, thyroid, thymus or heart.

HISTOPATHOLOGY (see attachment 4)

NON NEOPLASTIC
- Most histopathological changes observed were about equally distributed among the treatment groups and the controls and represented normal background pathology for rats of this strain and age. A number of treatment-related non-neoplastic changes were, however, observed. Dose-related increases in the incidence of zona glomerulosa hypertrophy occurred in all treatment groups in both sexes at the end of the 30-month study. The zona glomerulosa was distinctly wider than in controls; the cells in this area were enlarged and showed a finely vacuolar cytoplasm. Zona glomerulosa hypertrophy was not associated with increased plasma potassium concentration nor increased urinary potassium excretion. The hypertrophy of the zona glomerulosa observed with NH4Cl is ascribed to chronic stimulation of the adrenal cortex by NH4Cl induced acidosis.
- With 2.1 % NH4Cl, the incidence of oncocytic tubules was significantly decreased after 30 months. Oncocytic tubules do occur spontaneously in untreated rats and are a common observation in aged male rats with severe chronic progressive nephrosis. The overall incidence of nephrosis was comparable among the groups throughout the studies, but after 30 months the incidence of severe nephrosis was decreased in males of the 2.1 % NH4Cl group, while the qualification "very severe nephrosis" did not occur. Nephrosis or "chronic progressive nephrosis" is a common lesion in ageing male Wistar rats, and is characterised by loss of a progressive number of nephrons due to glomerularsclerosis and subsequent protein leakage. The decrease in incidence noted may suggest a protective effect of NH4Cl on the development of nephrosis. Surprisingly, however, granular casts in the urinary sediment, which may be indicative of renal disease, were noted especially in rats receiving 2.1 % NH4Cl.

NEOPLASTIC
- There were no treatment-related changes in any specific tumor type among the groups. In females, relatively high incidences of (adeno)carcinomas were found in the mammary gland with 2.1% NH4Cl, but because these changes were not accompanied by pre-neoplastic alterations in the 18- and 30-month studies and because their incidences were within the range of historical control data, they were not ascribed to treatment. The total number of rats with tumors or the total incidence of tumors was not affected by the treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

(30 months)

Effect level:

> 1 104.6 mg/kg bw/day (actual dose received)

Sex:

male/female

In summary, 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 to life-long exposure to the acid load. No adverse effects were noted with NH4Cl. It appeared that, in rats, NH4Cl-induced chronic metabolic acidosis was not associated with dissolution of alkaline bone salts. Finally, a protective effect of metabolic acidosis on tumour development was not found.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Principles of method if other than guideline:

The test substance is administered to a group of mice via drinking water on a daily basis for a duration of up to 652 days. At termination, histopathology of the bladder and major organs were performed. Urin pH was monitored in all animals.

GLP compliance:

no

Species:

mouse

Strain:

other: Hybrid (1F X C57/F1)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: in bred animals
- Age at study initiation: 10 - 12 weeks
- Diet: ad libitum; Oxoid diet
- Water: ad libitum

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

652 days

Frequency of treatment:

daily 7 days/week

Post exposure period:

no

Dose / conc.:

1 other: %

Remarks:

Basis: nominal in water

No. of animals per sex per dose:

50 animals

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

URINALYSIS: Yes
- Animals fasted: all
- Parameters checked: pH

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (bladder)
HISTOPATHOLOGY: Yes
- Organs: bladder and all major organs (no further details)

Details on results:

MORTALITY
- The average survival time was 492 days (control: 485 days)

HISTOPATHOLOGY: NEOPLASTIC
- Control: no tumours were found in the bladder, no epithelial hyperplasia, no calculi
- NH4Cl: no tumours were found in the bladder, mild epithelial hyperplasia 1/50, no calculi, nephritis in kidney, 1/50


URINALYSES
- Control: pH 6.2
- NH4Cl: pH 6.2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 other: %

Sex:

female

The average survival time was 492 days. (Control: 485 days). Control animals and those given ammonium chloride were free from bladder tumours, epithelial hyperplasia and calculi.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Principles of method if other than guideline:

F344 male and female rats were administered 1.0 % NH4Cl for 8 weeks and changes in the urinary bladder histopathology and the urinary components were examined.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Wako Pure Chemical

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc., Atsugi, Japan
- Age at study initiation: 6 weeks old
- Fasting period before study: no
- Housing: housed three to five per plastic cage on soft wood chip bedding
- Diet: ad libitum, commercial pelleted basal diet (Oriental M, Oriental Yeast Co., Tokyo, Japan)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- 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:

8 weeks

Frequency of treatment:

daily, 7 day/week

Post exposure period:

no

Dose / conc.:

1 other: %

Remarks:

nominal in diet

No. of animals per sex per dose:

6 male and 5 female rats

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: no
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

URINALYSIS: Yes
- Time schedule for collection of urine: during week 8, on three consecutive days, 6 hours/day
- Metabolism cages used for collection of urine: yes
- Animals fasted: Yes
- Parameters checked: pH (forced urination), volume, elctrolytes (sodium, potassium, calcium, chloride)

Sacrifice and pathology:

SACRIFICE:
- Time schedule: at the end of week 8

GROSS PATHOLOGY: Yes, bladder
HISTOPATHOLOGY: Yes, bladder

Statistics:

Statistical analyses were performed by using Student's t and Welch's t tests in combination with the F test for variability between means, and by the cummulative chi square method in 2 X 5 tables for analysis of proliferative lesions in the urinary bladder

Details on results:

CLINICAL SIGNS AND MORTALITY
- No animal died before termination of the study

BODY WEIGHT AND WEIGHT GAIN:
- Significantly lower than control, especially in male rats (263 - 281 vs. 313 g in controls) (values of treated group represent all males used in the study. In the same study, 5 other groups were included to test the effects of o-phenyphenol(OPP) and sodium o-phenyphenol(OPP) in combination with NH4Cl and NaHCO3) on bladder

FOOD CONSUMPTION AND COMPOUND INTAKE
- There were no significant differences in food intake between the treated group and the control group (individual animal data or mean values not given)

WATER CONSUMPTION
- Water consumption was comparable between the treated and control groups (individual animal data or mean values not given)

URINALYSES
- Urine volume: significantly (p < 0.05) increased in males compared to controls (5.7 vs. 4.0 mL/18h in controls for males and 4.5 vs. 4.1 mL/18h in controls for females)
- pH: significantly (p < 0.001) decreased in both sexes (5.91 vs. 7.0 in controls for males and 5.89 vs. 6.81 in controls for females)
- Osmolarity: significantly (p< 0.001) decreased compared to controls in males only (879 ± 290 vs. 1536 ± 3308 mOsm/kg H2O)
- Electrolytes: males; sodium (p < 0.001) and potassium (p < 0.01) were significantly reduced in males only while sodium concentration were significantly (p < 0.01) reduced in the females. Calcium contrentrations were significantly (p < 0.05) increased in male animals

HISTOPATHOLOGY
- Urinary bladder analysis: No hyperplasia and no tumours were noticed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 other: %

Sex:

male/female

Table 1: Urinalysis data for rats fed NH4Cl and non treatment controls (mean ± SD values)

		Na+	K+	Ca2+	Cl-
		mEq/liter
Male	Control	125 ± 9	197 ± 33	4.8 ± 1.3	201 ± 48
	NH4Cl	38 ± 23^a	117 ± 41^b	6.5 ± 0.8^c	206 ± 66
Female	Control	155 ± 35	143 ± 41	8.7 ± 2.9	167 ± 26
	NH4Cl	82 ± 30^b	128 ± 40	10.3 ± 4.3	216 ± 58

 

Significantly different from no treatment group at :^a, P < 0.001;^b, P < 0.01;^c, P<0.05

1) Body weight: significantly lower than control, especiallyin male rats.
2) Urine volume: increased in male
3) Urine pH: decreased in both sexes
4) Urinary bladder analysis: No hyperplasia (Positive control: 1.25 % o-phenyphenol(OPP) plus 3.0 % NaHCO3)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on carcinogenicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.

Additional information

In a 30 months feedings study, ammonium chloride (> 99.5% pure) was administered in diet continuously to fifty (5 week-old) Wistar rats/sex/dose at two doses: namely 1.0 % and 2.1 % for a duration of 30 months (ca. 131 weeks). The control group was presented non supplemented diets. No treatment-related abnormalities in condition or behaviour were observed in the rats of this study. The clinical effects noted were of random nature and corresponded to the usual ageing symptoms seen in this strain of rats. There were also no adverse compound related effects on mortality, food consumption, haematology, clinical chemistry, urinalysis, or organ weights. The type and incidence of palpable masses noted during the chronic studies did not indicate any treatment-related effects. Body weights were significantly reduced at various periods over the 30 months study period in females of the low dose group and both sexes of the high dose group. Histopathology examinations revealed dose-related increases in the incidence of zona glomerulosa hypertrophy in all treatment groups in both sexes at the end of the 30-months study. Zona glomerulosa was however not associated with increased blood potassium concentration or increased potassium excretion. Early increases (after 4 and 13 weeks) in zona glomerulosa hypertrophy were also noted with the high (4%) level of NH4Cl (see chapter 7.5.1). With 2.1% NH4Cl, the incidence of oncocytic tubules was significantly decreased after 30 months. The overall incidence of nephrosis was comparable among the groups throughout the studies, but after 30 months the incidence of severe nephrosis was decreased in males of the 2.1% NH4Cl group. The NOAEL for toxicity is 2.1% NH4Cl (1104.6 mg/kg bw).

At the doses tested, there were no treatment-related increases in the incidence or changes in any specific tumour type among the groups. The relatively high incidences of (adeno)carcinomas found in the mammary gland of females treated with 2.1% NH4Cl, were not considered treatment related because these changes were not accompanied by pre-neoplastic alterations in the 18- and 30-months studies and because their incidences were within the range of historical control data. The NOAEL for carcinogenicity is 2.1% (1104.6 mg/kg bw) NH4Cl in diet. This carcinogenicity study in the rats is acceptable and satisfies the guideline OECD 451 requirement for a carcinogenicity study in rats.

 

In a mouse study, treatment for 50 female 1F X C57/F1 mice/dose, 7 days/week for 652 days with 1.0 % ammonium chloride (no data on purity) in drinking water did not cause any tumours in the bladder. Neither was there any epithelial hyperplasia or calculli found in the bladder. The average survival time was 492 days (control: 485 days) (Flaks and Clayson, 1975).

The above data indicates no carcinogenic properties for ammonium chloride. Instead, ammonium chloride seems to possess anticarcinogenic potential. Lina et al (2004) proposed the reduction of systemic pH as a possible mechanism for the apparent anticarcinogenic potential of NH4Cl. In an initiation/promotion study reported by Higiwara (1994), the incidence of hyperplasia and papillomas were reduced in male Fisher 344 (DuCrj) rats (no data on age) after a 32 week administration via feed of 1.0 % ammonium chloride (no data on purity). Prior to treatment with ammonium chloride, the test animals were treated with 0.05 % (N-butyl-N-(4-hydroxybutyl) nitrosamine for 4 weeks. The control animals were given non supplemented diets after the 4 week tumour induction phase.