Potassium hydrogencarbonate

Endpoint summary

Description of key information

Reliable, adequate and relevant studies on oral repeated dose toxicity of potassium hydrogencarbonate are available. These studies included 4-weeks, 13-weeks, 18-months, and 30-months studies, in which rats were treated with very high doses of potassium hydrogencarbonate in their feed (2 and 4%). The studies were not performed according to an explicitly mentioned international guideline, but accomplishment and documentation cover a broad range of the requirements of the usual guidelines for repeated dose toxicity studies. Thus, the studies are relevant, reliable and adequate for the purpose of assessing repeated dose toxicity. Even the low dose level of this study set exceeds the guideline limit dose for repeated dose toxicity studies to a considerable degree, no treatment related toxic effects relevant to humans were seen.
The NOAELs of potassium hydrogencarbonate relevant to humans is the highest dose tested of 4 % in diet, based on body weight and diet intake corresponding to
- 6054 mg/kg bw/d (43.8 mmol/kg bw/d) in males and 6137 mg/kg bw/d (44.4 mmol/kg bw/d) in females in the 4-weeks-study
- 4326 mg/kg bw/d (31.3 mmol/kg bw/d) in males and 4879 mg/kg bw/d (35.3 mmol/kg bw/d) in females in the 13-weeks-study
- 2861 mg/kg bw/d (20.7 mmol/kg bw/d) in males and 3566 mg/kg bw/d (25.8 mmol/kg bw/d) in females in the 18-months study and of
- 2667 mg/kg bw/d (19.3 mmol/kg bw/d) in males and 3331 mg/kg bw/d (24.1 mmol/kg bw/d) in females in the 30-months study

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication report which meets basic scientific principles

Qualifier:

no guideline followed

Principles of method if other than guideline:

Non-guideline 13-week-study to examine the effects of died-induced acid-base disturbances on urinary bladder epithelium. Diets were supplemented with high amounts of a base-forming diet (diets supplemented with potassium hydrogencarbonate) or acid forming diets (diets supplemented with ammonium chloride. In a first experiment diets were supplemented with, 2.5% KHCO3, or monosodium glutamate with or without additional NaCO3 or NH4Cl. In a second experiment, the amounts of KHCO3 and NH4Cl added to diets increased gradually from 1% in wk 2 to 5% in wk 6 and was subsequently maintained at 5%. Results were compared to with results on other alkalizing agents (monosodium glutamate (6%) with or without additional NaHCO3 (1.6%) or NH4Cl (1%).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

- Strain: [Cpb: WU]; Wistar
- Source: TNO.Central Institute for the Breeding of Laboratory Animals, Zeist, NL
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: group-caged, four or five together, in stainless-steel cages with wire-screen bottom and front
- Diet: ad libitum
-- a cereal-based open formula diet called 'stock diet' (which has been used for many years in the Institute, as a rat diet for stock, breeding and experimental animals, without any major changes in composition
-- a 'purified' diet containing acid casein supplemented with methionine as the only source of protein
- Water: tap water ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 1
- Humidity (%): 40- 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
- a cereal-based open formula diet called 'stock diet' (which has been used for many years in the Institute, as a rat diet for stock, breeding and experimental animals, without any major changes in composition was supplemented with test items: 2.5% KHCO3 in a first experiment, in the second experiment amount supplemented to diet gradually increased from 1% in wk 2 to 5% in wk 6 and was subsequently maintained at 5%.
- and a 'purified' diet containing acid casein supplemented with methionine as the only source of protein was supplemented with test items (for KHCO3 not used in first experiment): in the second experiment amount supplemented to diet gradually increased from 1% in wk 2 to 5% in wk 6 and was subsequently maintained at 5%.
- no further date on diet preparation

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
2.5% in the diet
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
5 % in the diet
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, no further data

DETAILED CLINICAL OBSERVATIONS: Yes, no further data

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of the individual rats were generally recorded at the initiation of the studies and then in weekly intervals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, but food consumption per cage was measured over 1 week periods
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: The water intake of the rats in each cage was measured on working days during weeks 3. 6, 8 and 11

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: at different stages of the studies, samples of urine were collected routinely from the groups of rats in each cage during the first 2 hr following the beginning of the light period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No, only during urine collection, food and drinking water were withheld
- Parameters examined: pH, volume, acid indices of the urine (total titratable acid, bicarbonate ion concentration and ammonium concentration were measured; net acid  excretion was calculated as the sum of total titratable acid plus ammonium ion concentration minus bicarbonate ion concentration)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, Microscopic examinations were restricted mainly to the urinary system. The bladder was fixed by injection of a measured volume of Bouin´s fixative. Tissue samples of the other organs examined (ureters, kidneys, liver, testes, thyroid with parathyroid, adrenals and bone) were fixed in 4% aqueous neutral phosphate-buffered formaline solution. Samples of the fixed organs were embedded in paraffin wax, sectioned at 5 µm and stained with hematoxylin and eosin. The bladder was sectioned at two levels to enable examinations of the rostral and caudal parts. The kidneys were examined in one transverse section and one longitudinal section.

Statistics:

no data

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):

effects observed, treatment-related

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- Mortality: only one animal in the NH4Cl-group died
- Clinical signs: no abnormalities in condition or behaviour

BODY WEIGHT AND WEIGHT GAIN
- stock diet supplemented with 2.5% or 5% KHCO3: no significant effect
- purified casein diet supplemented with 5% KHCO3: growth retardation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- no significant effects in KHCO3-groups

FOOD EFFICIENCY:
- no data

WATER CONSUMPTION:
no significant effects in KHCO3-groups

OPHTHALMOSCOPIC EXAMINATION:
- not performed

HAEMATOLOGY
- not perforemd

CLINICAL CHEMISTRY
- not performed

URINALYSIS
- pH: mean values of 8.44 in first experiment (2.5% dose); values around 8.5 in both KHCO3-groups (5% doses) in second experiment; in stock diet control  group 7.0 during the first 8 weeks with onward trend during the remaining 5 weeks; in purified casein supplemented diet control group  around 6.0
- Bicarbonate ion concentration (mequiv/l) (not measured in first experiment):
-- stock diet supplemented with 5% KHCO3: 75.5 (concurrent control  group: 22.0)
-- purified casein diet supplemented with 5% KHCO3: 62.5 (concurrent control  group: 12.0)
- Total titratable acid (mequiv/l):
-- stock diet supplemented with 5% KHCO3: - 33.5 (concurrent control  group: 12.0)
-- purified casein diet supplemented with 5% KHCO3: - 41.0 (concurrent control  group: 101.0)
- Ammonium ion concentration (mequiv/l):
-- stock diet supplemented with 5% KHCO3: 24.5 (concurrent control  group: 77.5)
-- purified casein diet supplemented with 5% KHCO3: 40.5 (concurrent control  group: 219.0)
- Net acid concentration (mequiv/l):
-- stock diet supplemented with 5% KHCO3: - 84.5 (concurrent control  group: 67.5)
-- purified casein diet supplemented with 5%: - 63.0 (concurrent control  group: 308.0)

NEUROBEHAVIOUR
not performed

ORGAN WEIGHTS
- Organ weights: relative kidney weights in all KHCO3-groups increased;  relative liver weights unaffected; no further organ weights reported

GROSS PATHOLOGY
not reported

HISTOPATHOLOGY: NON-NEOPLASTIC
-Urinary bladder: 
-- Minimal and slight focal epithelial hyperplasia: not significant in all KHCO3-groups
-- Minimal and severe diffuse epithelial hyperplasia: not significant in  both KHCO3-groups (5% doses) in second experiment (no data on first experiment, 2.5% dose)
-- Moderate diffuse epithelial hyperplasia: significant in all  KHCO3-groups (7/10 in first experiment (2.5% dose), 6/10 in both groups (5% doses) in second experiment, concurrent control groups 0/10)
-- Intra-epithelial cysts:
--- stock diet supplemented with 2.5% KHCO3 (first experiment): significant (6/10, concurrent  control group: 0/10)
--- stock diet supplemented with 5% KHCO3 (second experiment): significant (8/10, concurrent  control group: 0/10)
--- purified casein diet supplemented with 5% (second experiment): significant (6/10,  concurrent control group: 0/10)
- Further examined tissues and organs: no treatment-related changes
SATELITTE GROUP MAINTAINED ON monosodium glutamate with or without additional NaCO3: comparable effects to dose grops maintained on KHCO3 supplemented diets
SATELITTE GROUP MAINTAINED ON 5% NH4Cl: Urinary pH decreased; titratable  acid, ammonium ion and net acid concentration increased; not significant  hyperplasia of the urinary bladder epithelium

HISTOPATHOLOGY: NEOPLASTIC
- no effects

HISTORICAL CONTROL DATA
- no data

OTHER FINDINGS
none

Critical effects observed:

no

Feeding of KHCO3 (5%) or monosodium glutamate (6%) with or without NaHCO3 (1.6%) to rats for 13 weeks resulted in moderate hyperplasia of the bladder epithelium; these effects were accompanied by pH values of the urine > 8; simultaneous feeding of the acidifying salt NH4Cl to monosodium glutamate prevented the epithelial response and alkalization of urine; feeding of a diet containing NH4Cl alone resulted in moderate to severe hyperplasia of the bladder epithelium, accompanied by an acidified urine. According to the discussion by the study authors, these findings indicate that the bladder changes induced by KHCO3 are attributable to its alkalizing properties rather than to KHCO3 per se.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Non-guideline 13-week-study to examine the effects of died-induced acid-base disturbances. Diets were supplemented with high amounts of potassium hydrogencarbonate (2% or 4%, base-forming diets), ammonium chloride (2.1% or 4%, acid forming diet), or potassium chloride (3%, neutral diet providing K+ and Cl- in amounts equimolar to those in the 4% potassium hydrogencarbonate diet and the 2.1% ammonium chloride diet, respectively).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: SPF CpB:WU (Wistar random)
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands
- Age at study initiation:  about 5 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: in 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, Institute's cereal-based open formula diet
- Water: ad libitum, tap water
- Acclimation period: no data

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

Details on oral exposure:

DIET PREPARATION
- Institute's cereal-based open formula diet was supplemented with test items
- no further date on diet preparation

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

2 other: % in diet

Dose / conc.:

4 other: % in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Study groups:
- control group: unsupplemented institutes cereal based rodent diet
- 2% potassium hydrogencarbonate in diet (approximately 2046 mg/kg bw/d for males and 2294 mg/kg bw/d for females)
- 4% potassium hydrogencarbonate in diet (approximately 4326 mg/kg bw/d for males and 4879 mg/kg bw/d for females)
- Satellite groups: 2.1% and 4% NH4Cl (acid-forming diets) and neutral diet supplemented with 3% KCl providing K+ and Cl- in amounts equimolar to those in the 4% KHCO3 diet and the 2.1% NH4Cl diet, respectively
Post-exposure period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, measured weekly per cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, calculated per cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily per cage in weeks 1, 4, 6, 8, and 12 (24 hours consumption)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4 and 13 (tail tip blood)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined: haemoglobin concentration, packed cell volume, red blood cells, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thrombocytes, total white blood cells, prothrombin time, and differential white blood cells

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 5 (for blood gas analysis), week 13 (clinical chemistry parameters), week 4 and 12 (glucose analysis)
- Animals fasted: No data (except on glucose analysis)
- How many animals: all
- Parameters examined.:
--Blood gas analysis (tail tip blood): determination of pH, pCO2 and pO2 directly after sampling in capillary tubes, and calculation of bicarbonate and base excess
-- Clinical chemistry:
--- Plasma from abdominal aorta collected at necropsy: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, lactate dehydrogenase, total protein, albumin, total bilirubin, urea, creatinine, calcium, inorganic phosphate, chloride, sodium, and potassium
--- Whole blood collected from the tip of the tail in weeks 4 and 12 after overnight fasting: glucose

URINALYSIS: Yes (Urinary acid indices)
- Time schedule for collection of urine: weeks 1, 4, 6, and 10
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food and water for 2-3 h at the start of the light period)
- How many animals: all
- Parameters examined in fasted animals: urinary pH was measured in individual samples, mean pH values were calculated as the negative logarithm of the mean of the hydrogen concentrations instead of as the arithmetical mean of the individual pH values; bicarbonate, titratable acid, ammonia, and urea were measured in pooled samples (two samples per group and sex) and related to creatinine

URINALYSIS: Yes (standard urine parameter)
- Time schedule for collection of urine: week 6
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: all
- Parameters examined: volume (calibrated tubes), density, gamma glutamyl transferase, creatinine, urea, calcium, potassium, sodium, phosphate, and sulphate

URINALYSIS: Yes (hydroxyprolin)
- Time schedule for collection of urine: week 11
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: all (two samples from five rats per group and sex)
- Parameter examined: hydroxyproline

URINALYSIS: Yes (Concentrating ability of the kidneys)
- Time schedule for collection of urine: weeks 4, 7, and 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, urine collected during the last 16 h of a 24-h water deprivation period, during the 16-h collection period no feed was available
- How many animals: all
- Parameters examined: volume (graduated tubes), density, and 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

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- CALCIUM CONTENT in FEMUR: week 13 at autopsy, the right femur from 10 animals per group and sex was removed, cleaned from adherent soft tissue and weighed, dried at 100 °C, subjected to fat extraction (petroleum ether), redryed and the weight recorded as fat free solid, the fat free femur was ashed at 550 °C and the calcium content was determined by atomic absorption spectrophotometry

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- end of week 13, all rats were killed on a number of successive working days by exsanguination from the abdominal aorta, under light ether anaesthesia, and subjected to a thorough post-mortem examination. Any abnormalities were recorded and the adrenals, brain, pituitary, kidneys, liver, spleen, testes, ovaries, thyroid, thymus, and heart were weight. Paired organs were weighed together

HISTOPATHOLOGY: Yes
- 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. The lungs and urinary bladder were inflated with the fixative. Paraffin-embedded, 5 µm sections of the above tissues from all animals of each sex in the control group, the potassium hydrocarbon and the ammonium chloride high-dose group were stained with haematoxylin and eosin and examined microscopically. Adrenals, heart, kidneys, stomach, thyroid, testes, urinary bladder, and uterus were also examined in the intermediate-dose groups

Other examinations:

no

Statistics:

Numerical data were evaluated for statistical significance using one-way analysis of (co)variance [AN(CO)OVA] followed by Dunnett's test, 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

Description (incidence and severity):

- no treatment-related abnormalities in condition or behaviour; there was only the usual random incidence of ageing symptoms that occur in this strain of rats when maintained over a period of time

Mortality:

no mortality observed

Description (incidence):

- Mortality and time of death: no animal died

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no treatment-related effects

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Food consumption: no treatment-related effects
- ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX:
-- males: 2046 and 4326 mg/kg bw/d (14.8 and 31.3 mmol/kg bw/d)
-- females: 2294 and 4879 mg/kg bw/d (16.6 and 35.3 mmol/kg bw/d)
(recalculation of dose from data given in mmol/kg bw/d (molecular weight=138.21 mg/mmol) )

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

- in the 2% and the 4% dose group approximately 10% and 40% increased, respectively

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- no consistent or treatment-related effects on red blood cell variables, clotting potential or total and differential white blood cell counts

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Blood gas analysis: dose-related increase in base excess, associated with higher blood pH and bicarbonate concentrations
- Clinical biochemistry: potassium levels in plasma were generally increased in males and females fed 2 or 4% potassium hydrogencarbonate, although the differences with the controls were not always statistically significant; significant base excess in both sexes of both dose groups; no treatment-related effects on further measured blood clinical biochemistry parameters

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Urine parameter of non-fasted animals: urine volume increased in both sexes of the high dose group; the urinary density did not show consistent differences but tended to be decreased in males of the high dose group; potassium excretion increased in both sexes in both dose groups; urinary sodium excretion tended to be relatively high at various occasions in rats fed 4% potassium hydrogencarbonate, which may be ascribed to the natriuretic effect of high potassium intake but the figures showed large variations; no treatment related effects on excretion of calcium, phosphate, sulphate, gamma glutamyl transferase or hydroxyproline
- Urinary pH and acid indices: pH increased in both dose groups, net acid excretion (= ammonium ion (NH4+) excretion + urinary titratable acidity - bicarbonate excretion) considerable decreased in both sexes of the high dose group, and to a lesser extend in the low dose group
- Concentrating ability of the kidneys: the renal concentration test (no food and water available prior to and during urine sampling) showed a slightly increased volume associated with a somewhat decreased density of the urine in males of both dose groups; brownish discoloration of the urine and haematuria, as detected with urinary test stripes and by microscopic examination of the urinary sediment were occasionally increased, but there were no consistent or dose-related differences in incidence or severity of haematuria among the groups; the occurrence of crystals was not affected in any group

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Kidney weights: relative kidney weights in females of the high dose group significantly increased and in males of this group tendentiously but not statistically significant increased
- Further examined organ weights: there were no consistent or treatment-related changes in the weights of adrenals, brain, testes, liver, spleen, ovaries, pituitary, thyroid, thymus or heart

Gross pathological findings:

no effects observed

Description (incidence and severity):

- macroscopic examination at necropsy did not reveal significant differences among the treatment groups and the controls

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Adrenals: in both sexes of the high dose group, the zona glomerosa was distinctly wider than in controls, the cells in this area were enlarged and showed a finely vacuolar cytoplasm (10 animals per sex examined, findings in 9/10 males (significant, control: 1/10) and 4/10 females (not significant, control: 0/10). This microscopic finding was interpreted by the study authors as adaptive response to chronic simulation of the adrenal cortex by potassium cations.
- Kidneys: in both sexes of the high dose group, oncocytic tubules but no severe or very severe nephrosis or urothelial hyperplasia in the pelvis (10 animals per sex examined, findings in 10/10 males (significant, control: 0/10) and 9/10 females (significant, control: 0/10). The oncocytic tubules were characterised by tubules lined with, often hypertrophy, epithelial cells containing eosinophilic granular cytoplasm (oncocytes), often showing a cystically dilated lumen with epithelial cells protruding into the lumen. As discussed by the study authors, oncocytic tubules do occur spontaneously in untreated rat and are commonly observed in aged males, regarded as regenerative hyperplasia or as a functional tubular hyperplasia in order to meet increased work load. The authors suggested that the hydrogen carbonate anion mainly determined the increased occurrence of this lesion.
- Urinary bladder: no treatment-related significant effects; examination of the urinary bladder showed (not significant) very slight to slight simple epithelial hyperplasia in 4/10 and 3/10 males (control: 0/10) and 1/10 and 1/10 females (control: 0/10) in the low and the high dose groups, respectively.
- Reproductive organs: no treatment-related effects (epididymides, testes, ovaries, and uterus examined)
- Further examined organs and tissues: no treatment-related effects

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- Calcium content in femur: no treatment-related effects

Dose descriptor:

NOAEL

Effect level:

4 326 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Highest dose tested (4% in diet); no adverse effects relevant to humans observed; only adaptive effects on very high ion ingestion seen.

Dose descriptor:

NOAEL

Effect level:

4 879 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: Highest dose tested (4% in diet); no adverse effects relevant to humans observed; only adaptive effects on very high ion ingestion seen.

Critical effects observed:

no

The results of this 13-week-study have been published together with the results of a 4-week-study, a 78-week(18 month)-study, and a 130-week(30 month)-study. The 4-week- and the 13-week-study were separate experiments, the 78-week- and the 130-week-study were started simultaneously. It is not always quite clear, whether the reported results e.g. for urine parameters are the results of a specific study or the summed up results of different studies.

Executive summary:

In a 13 -week toxicity study potassium hydrogencarbonate (a.i. >99.5%) was administered to 10 Wistar rats, strain SPF Cpb:WU/sex/dose in the diet at dose levels of 0, 2 and 4% (0, 2046 and 4326 mg/kg bw/d (14.8 and 31.3 mmol/kg bw/d) in males, respectively and 0, 2294 and 4879 mg/kg bw/d (16.6 and 35.3 mmol/kg bw/d) in females, respectively, based on body weight and food consumption.

Object of the study was to examine the effects of died-induced acid-base disturbances. Albeit the study was not performed according to a explicit mentioned international guideline, accomplishment and documentation cover a broad range of the requirements of the usual guidelines for repeated dose toxicity studies. The dose level exceeds the guideline limit dose for repeated dose toxicity studies which is 1000 mg/kg bw/d by a factor of almost 2 and of more than 4 in the low and high dose group, respectively.

The rats adapted relatively easily to the feeding of these very high doses and were free of treatment related adverse effects. The few treatment related changes seen are mostly an expression of the physiological adaptation to the very high ion intake and regarded as of no toxicological relevance.

The NOAEL is the highest dose tested of 4 % in diet, based on body weight and diet intake corresponding to 4326 mg/kg bw/d (31.3 mmol/kg bw/d) in males and 4879 mg/kg bw/d (35.3 mmol/kg bw/d) in females.

The study has been part of a study set comprising of this 13 -week study, a 4 -week study, a 18-month, and a 30-month study. The dose levels in all studies were 2 and 4% in diet. A summarizing discussion of the results of the whole study set is given in the endpoint summary on repeated dose toxicity.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Non-guideline 18-month-study to examine the effects of died-induced acid-base disturbances. Diets were supplemented with high amounts of potassium hydrogencarbonate (2% or 4%, base-forming diets), ammonium chloride (2.1% acid forming diet) or potassium chloride (3%, neutral diet, providing K+ and Cl- in amounts equimolar to those in the 4% potassium hydrogencarbonate and the 2.1% ammonium chloride diet, respectively).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: SPF CpB:WU (Wistar random)
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands
- Age at study initiation:  about 5 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: in 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, Institute's cereal-based open formula diet
- Water: ad libitum, tap water
- Acclimation period: no data

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

Details on oral exposure:

DIET PREPARATION
- Institute's cereal-based open formula diet was supplemented with test items
- no further date on diet preparation

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

78 weeks (18 months)

Frequency of treatment:

daily

Dose / conc.:

2 other: % in diet

Dose / conc.:

4 other: % in diet

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

Study groups:
- control group: unsupplemented institutes cereal based rodent diet
- 2% potassium hydrogencarbonate in diet (approximately 1299 mg/kg bw/d for males and 1686 mg/kg bw/d for females)
- 4% potassium hydrogencarbonate in diet (approximately 2861 mg/kg bw/d for males and 3566 mg/kg bw/d for females)
- Satellite groups: 2.1% NH4Cl (acid-forming diet) and neutral diet supplemented with 3% KCl providing K+ and Cl- in amounts equimolar to those in the 4% KHCO3 diet and the 2.1% NH4Cl diet, respectively
Post-exposure period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, including evaluation regarding grossly visible or palpable masses at regular intervals

BODY WEIGHT: Yes
- Time schedule for examinations: weekly or (from 3 months) once every 4 weeks

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, measured weekly or (from 3 months) once every 4 weeks per cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, calculated per cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes,
- Time schedule for examinations: daily per cage in weeks 1, 4, 6, 8, 12, 55, and 75 (24 hours consumption)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4, 13, 32, 55, and 75 (tail tip blood)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 per group and sex
- Parameters examined: haemoglobin concentration, packed cell volume, red blood cells, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thrombocytes, total white blood cells, prothrombin time, and differential white blood cells

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 5, 14, 32, 56, and 76 (for blood gas analysis), weeks 17 and 38 and month 18 (clinical chemistry parameters), weeks 4, 12, 36, and 77 (glucose analysis)
- Animals fasted: No data (except on glucose analysis)
- How many animals: 10 per group and sex
- Parameters examined.:
--Blood gas analysis (tail tip blood): determination of pH, pCO2 and pO2 directly after sampling in capillary tubes, and calculation of bicarbonate and base excess
-- Clinical chemistry:
--- month 18, plasma from the abdominal aorta obtained at necropsy, and in weeks 17 and 38 in samples obtained by orbita puncture: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, lactate dehydrogenase, total protein, albumin, total bilirubin, urea, creatinine, calcium, inorganic phosphate, chloride, sodium, and potassium
--- Whole blood collected from the tip of the tail of 10 animals per group and sex in weeks 4, 12, 36, and 77 after overnight fasting: glucose

URINALYSIS: Yes (Urinary acid indices)
- Time schedule for collection of urine: weeks 1, 4, 6, 10, 14, 36, 57, and 76
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food and water for 2-3 h at the start of the light period)
- How many animals: all
- Parameters examined in fasted animals: urinary pH was measured in individual samples, mean pH values were calculated as the negative logarithm of the mean of the hydrogen concentrations instead of as the arithmetical mean of the individual pH values; bicarbonate, titratable acid, ammonia, and urea were measured in pooled samples (two samples per group and sex) and related to creatinine

URINALYSIS: Yes (standard urine parameter)
- Time schedule for collection of urine: weeks 6, 16, 38, and 78
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: 10 per group and sex
- Parameters examined: volume (calibrated tubes), density, gamma glutamyl transferase, creatinine, urea, calcium, potassium, sodium, phosphate, and sulphate

URINALYSIS: Yes (hydroxyprolin)
- Time schedule for collection of urine: weeks 11 and 76
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: 10 per group and sex (two samples from five rats per group and sex)
- Parameter examined: hydroxyproline

URINALYSIS: Yes (Concentrating ability of the kidneys)
- Time schedule for collection of urine: weeks 4, 7, 13, 14, 36, 57, and 77
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, urine collected during the last 16 h of a 24-h water deprivation period, during the 16-h collection period no feed was available
- Parameters examined: volume (graduated tubes), density, and 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, and 77

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- CALCIUM CONTENT in FEMUR: month 18 at autopsy, the right femur from 10 animals per group and sex was removed, cleaned from adherent soft tissue and weighed, dried at 100 °C, subjected to fat extraction (petroleum ether), redryed and the weight recorded as fat free solid, the fat free femur was ashed at 550 °C and the calcium content was determined by atomic absorption spectrophotometry
- CALCIUM and PHOSPHORUS EXCRETION in URINE and FAECES: In week 53, six rats per sex and 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- end of month 18, all rats were killed on a number of successive working days by exsanguination from the abdominal aorta, under light ether anaesthesia, and subjected to a thorough post-mortem examination. Any abnormalities were recorded and the adrenals, brain, pituitary, kidneys, liver, spleen, testes, ovaries, thyroid, and heart were weight. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
- The following tissues were preserved in 10% neutral phosphate buffered formalin: adrenals, aorta, axillary lymph nodes, brain, caecum, coagulating gland, colon, epididymides, exorbital lachrimal gland, eyes, heart, kidneys, liver, lungs, mammary gland, mesenteric lymph nodes, oesophagus, ovaries, pancreas, parathyroids, parotic salivary glands, pituitary, preputial/clitoral glands, prostate, rectum, seminal vesicles, skeletal muscle, skin, small intestine (duodenum, ileum, jejunum), spleen, sternum with bone marrow, stomach, submaxillary salivary glands, testes, thymus, thyroid, urinary bladder, uterus, and all gross lesions. The lungs and urinary bladder were inflated with the fixative. Paraffin-embedded, 5 µm sections of the above tissues from all animals of each sex in the control group, the potassium hydrocarbon and the ammonium chloride high-dose group were stained with haematoxylin and eosin and examined microscopically. Adrenals, heart, kidneys, stomach, thyroid, testes, urinary bladder, and uterus were also examined in the intermediate-dose groups.

Other examinations:

no

Statistics:

Numerical data were evaluated for statistical significance using one-way analysis of (co)variance [AN(CO)OVA] followed by Dunnett's test, 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

Description (incidence and severity):

- Clinical signs: no treatment-related abnormalities in condition or behaviour; there was only the usual random incidence of ageing symptoms that occur in this strain of rats when maintained over a period of time
- Grossly visible or palpable masses: no treatment-related effects

Mortality:

no mortality observed

Description (incidence):

- Mortality and time of death: mortality rate was not affected

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- mean body weights decreased in males of the high dose group (decrease in the range of 4 to 10%, no exact data given)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Food consumption: no treatment-related effects
- ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX:
-- males: 1299 and 2861 mg/kg bw/d (9.4 and 20.7 mmol/kg bw/d)
-- females: 1686 and 3566 mg/kg bw/d (12.2 and 25.8 mmol/kg bw/d)
(recalculation of dose from data given in mmol/kg bw/d (molecular weight=138.21 mg/mmol) )

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

- in the 2% and the 4% dose group approximately 10% and 40% increased, respectively

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- no consistent or treatment-related effects on red blood cell variables, clotting potential or total and differential white blood cell counts

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Blood gas analysis: dose-related increase in base excess, associated with higher blood pH and bicarbonate concentrations
- Clinical biochemistry:potassium levels in plasma were generally increased in males and females fed 2 or 4% potassium hydrogencarbonate, although the differences with the controls were not always statistically significant; significant base excess in both sexes of both dose groups; no treatment-related effects on further measured blood clinical biochemistry parameters

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Urine parameter of non-fasted animals: urine volume increased in both sexes of the high dose group, 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 the high dose group; potassium excretion increased in both sexes in both dose groups; urinary sodium excretion tended to be relatively high at various occasions in rats fed 4% potassium hydrogencarbonate, which may be ascribed to the natriuretic effect of high potassium intake but the figures showed large variations; no treatment related effects on excretion of calcium, phosphate, sulphate, gamma glutamyl transferase or hydroxyproline
- Urinary pH and acid indices: pH increased in both dose groups, net acid excretion (= ammonium ion (NH4+) excretion + urinary titratable acidity - bicarbonate excretion) considerable decreased in both sexes of the high dose group, and to a lesser extend in the low dose group
- Concentrating ability of the kidneys: the renal concentration test (no food and water available prior to and during urine sampling) showed a slightly increased volume associated with a somewhat decreased density of the urine in males of both dose groups and in females of the high dose group in week 77; brownish discoloration of the urine and haematuria, as detected with urinary test stripes and by microscopic examination of the urinary sediment were occasionally increased, but there were no consistent or dose-related differences in incidence or severity of haematuria among the groups; the occurrence of crystals was not affected in any group

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Kidney weights: relative kidney weights in both sexes of both dose groups tendentiously but except of low dose female group not statistically significant increased
- Further examined organ weights: there were no consistent or treatment-related changes in the weights of adrenals, brain, testes, liver, spleen, ovaries, pituitary, thyroid, or heart

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Macroscopic examination of the urine bladder: some animals showed thickening and/or induration of the urinary bladder wall, irregular serosal surface and/or luminal dilatation; on male of the 4% group was found to have a grossly visible bladder mass, which extended into the dorsal prostate; bladder stones were not observed
- Macroscopic examination of further organs and tissues: no significant differences among the treatment groups and the controls

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Adrenals: in both sexes of the high dose group, the zona glomerosa was distinctly wider than in controls, the cells in this area were enlarged and showed a finely vacuolar cytoplasm (13-15 animals per sex and dose examined (both survivors and intercurrent deaths), findings in the low and high dose group in 3 (not significant) and 10 males (significant, control: 0), respectively, and 2 (not significant) and 9 females (significant, control: 0), respectively. This microscopic finding was interpreted by the study authors as adaptive response to chronic simulation of the adrenal cortex by potassium cations.
- Kidneys: in both sexes of both dose groups, oncocytic tubules but no treatment-related significant severe or very severe nephrosis or urothelial hyperplasia in the pelvis (13-15 animals per sex and dose examined (both survivors and intercurrent deaths), findings in the low and high dose group in 4 (significant) and 15 males (significant, control: 0), respectively, and 5 (significant) and 13 females (significant, control: 0), respectively. The oncocytic tubules were characterised by tubules lined with, often hypertrophy, epithelial cells containing eosinophilic granular cytoplasm (oncocytes), often showing a cystically dilated lumen with epithelial cells protruding into the lumen. As discussed by the study authors, oncocytic tubules do occur spontaneously in untreated rat and are commonly observed in aged males, regarded as regenerative hyperplasia or as a functional tubular hyperplasia in order to meet increased work load. The authors suggested that the hydrogen carbonate anion mainly determined the increased occurrence of this lesion.
- Urinary bladder: in both sexes of both dose groups, the incidences of simple epithelial hyperplasia, of papillary hyperplasia and nodular hyperplasia were increased. However significantly and dose depended increases were limited to simple epithelial hyperplasie in both males and females of the low and the high dose group and papillary epithelial hyperplasia, nodular epithelial hyperplasia and transitional-cell papilloma (one or multiple) in the high dose females.
--Simple epithelial hyperplasia in 0/15, 5/13 (significant) and 6/15 (significant) males, and 1/15, 5/15 (not significant) and 9/15 (significant) females in the control, the low- and the high-dose group, respectively
-- Papillary epithelial hyperplasia in 0/15, 4/13 (significant) and 1/15 (not significant) males, and 0/15, 1/15 (not significant) and 5/15 (significant) females in the control, the low- and the high-dose group, respectively
-- Nodular epithelial hyperplasia in 0/15, 2/13 (not significant) and 0/15 (not significant) males, and 0/15, 1/15 (not significant) and 4/15 (not significant) females in the control, the low- and the high-dose group, respectively
-Reproductive organs: no treatment-related effects (coagulating gland, epididymides, prostate, seminal vesicles, testes, mammary gland, ovaries, uterus, and preputial/clitoral glands examined)
-Further examined organs and tissues: no treatment-related effects

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Urinary bladder:
-- Transitional-cell papilloma (one or multiple) in 0/15, 0/13 (not significant) and 0/15 (not significant) males, and 0/15, 0/15 (not significant) and 2/15 (not significant) females in the control, the low- and the high-dose group, respectively
-- Transitional-cell carcinoma in 0/15, 0/13 (not significant) and 1/15 (not significant) males, and 0/15, 0/15 (not significant) and 2/15 (not significant) females in the control, the low- and the high-dose group, respectively
- Total tumour incidence: Apart from preneoplastic and neoplastic lesions in the urinary bladder, there were no treatment-related changes in any specific tumor type among the groups; potassium hydrogencarbonate did neither affect type, incidence and multiplicity of tumours, nor time of tumor appearance and the ratio benign-malignant tumours.

HISTORICAL CONTROL DATA
- Neoplastic alterations of urinary bladder: Historical control data obtained in 18 different chronic (exact duration not mentioned) studies with Wistar rats of the laboratory revealed no papillomas or carcinomas of the urinary bladder in 795 male and 777 female control rats.

Other effects:

no effects observed

Description (incidence and severity):

- Calcium content in femur: no treatment-related effects
- Excretion of calcium and phosphorus in faeces and urine: no treatment-related effects

Dose descriptor:

NOAEL

Effect level:

2 861 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

3 566 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

In the urinary bladder in both sexes of both dose groups, the incidence of simple epithelial hyperplasia, of papillary hyperplasia and nodular hyperplasia were increased. However in males except of simple epithelial hyperplasia, the increases were not dose depended and in females except of simple epithelial hyperplasia the increase was only significant in high dose group. These findings are consistent with a known nongenotoxic mechanism of bladder carcinogenesis in the rat.

The relevance to humans of preneoplastic or neoplastic urinary bladder findings in rats induced by high dose alkali intakes (e.g. as counterions in food additives like artificial sweetener or flavour enhancer) and associated by alkalinization of the urine, elevated urine volumes, altered urine electrolytes composition with or without renal pelvic mineralization or urine precipitation which leads to a cyctotoxic effect on the bladder epithelium with consequent regenerative proliferation and ultimately tumors have been discussed for a long time (e.g. by the International Agency for Research on Cancer as part of the World Health Organization (IARC) in the IARC Monography Vol. 73 (1999) and in the IARC Scientific Publication No 147 (1999) or more recently by the European Food Safety Authority (EFSA) in the option on Aspartam (2006) or a review by Cohen, SM (2008)). It is widely accepted that these high dose effects are specific to the rat and are of no relevance to humans.

The results of this 78-week-study have been published together with the results of a 4-week-study, a 13-week-study, and a 130-week (30 month)-study. The 4-week- and the 13-week-study were separate experiments, the 78-week- and the 130-week-study were started simultaneously. It is not always quite clear, whether the reported results e.g. for urine parameters are the results of a specific study or the summed up results of different studies.

Furthermore, is not quite clear, whether specific parameters (e.g. haematological or urine parameters) were measured until completion of the 78-week-study parallel in two separate groups per sex and dose in the 78-week- and the 130-week-study-groups or only in one group per sex and dose used for both studies.

Quoted references:

Cohen, SM: Review - Thresholds in Genotoxicity and Carcinogenicity: Urinary Bladder Carcinogenicity, Genes and Environment, 40(4), 132 -138 (2008)

European Food Safety Authority (EFSA): Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC) on a request from the Commission related to a new long-term carcinogenicity study on aspartame Question number EFSA-Q-2005-122 Adopted on 3 May 2006, The EFSA Journal 356, 1-44 (2006)

World Health Organization International Agency for Research on Cancer (IARC): Some Chemicals that Cause Tumours of the Kidney or Urinary Bladder in Rodents, and Some Other Substances, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Vol. 73 (1999)

World Health Organization International Agency for Research on Cancer (IARC): Species Differences in Thyroid, Kidney and Urinary Bladder Carcinogenesis, IARC Scientific Publication, No 147 (1999)

Executive summary:

In a 78 -week (18-month) toxicity study potassium hydrogencarbonate (a.i. >99.5%) was administered to 15 Wistar rats, strain SPF Cpb:WU/sex/dose in the diet at dose levels of 0, 2 and 4% (0, 1299 and 2861 mg/kg bw/d (9.4 and 20.7 mmol/kg bw/d) in males, respectively and 0, 1686 and 3566 mg/kg bw/d (12.2 and 25.8 mmol/kg bw/d) in females, respectively, based on body weight and food consumption.

Object of the study was to examine the effects of died-induced acid-base disturbances. Albeit the study was not performed according to a explicit mentioned international guideline, accomplishment and documentation cover a broad range of the requirements of the usual guidelines for repeated dose toxicity studies. The dose level exceeds the guideline limit dose for repeated dose toxicity studies which is 1000 mg/kg bw/d in the low dose group by almost 30 or 70 % and in the high dose group by almost 190 or 260 % for males and females, respectively.

The rats adapted relatively easily to the feeding of these very high doses and were free of treatment related adverse effects relevant to humans. Most treatment related changes seen are an expression of the physiological adaptation to the very high ion intake and regarded as of no toxicological relevance. In the urinary bladder significant preneoplastic histopathological epitelial alterations have developed, which are common findings in rats after long term high dose alkali intakes. The relevance to humans of preneoplastic or neoplastic urinary bladder findings in rats induced by high dose alkali intakes (e.g. as counterions in food additives like artificial sweetener or flavour enhancer) and associated by alkalinization of the urine, elevated urine volumes, altered urine electrolytes composition with or without renal pelvic mineralization or urine precipitation which leads to a cyctotoxic effect on the bladder epithelium with consequent regenerative proliferation and ultimately tumors have been discussed for a long time (e.g. by the International Agency for Research on Cancer as part of the World Health Organization (IARC) in the IARC Monography Vol. 73 (1999) and in the IARC Scientific Publication No 147 (1999) or more recently by the European Food Safety Authority (EFSA) in the option on Aspartam (2006) or a review by Cohen, SM on Thresholds in Genotoxicity and Carcinogenicty: Urinary Bladder Carcinogenisis (2008)). It is widely accepted that these high dose effects are specific to the rat and are of no relevance to humans.

The NOAELrelevant to humans is the highest dose tested of 4 % in diet, based on body weight and diet intake corresponding to 2861 mg/kg bw/d (20.7 mmol/kg bw/d) in males and 3566 mg/kg bw/d (25.8 mmol/kg bw/d) in females.

The study has been part of a study set comprising of this 78 -week (18 month) study, a 4 -week study, a 13 -week study and a 30-month study. The dose levels in all studies were 2 and 4% in diet. A summarizing discussion of the results of the whole study set is given in the endpoint summary on repeated dose toxicity.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Non-guideline 30-month-study to examine the effects of died-induced acid-base disturbances. Diets were supplemented with high amounts of potassium hydrogencarbonate (2% or 4%, base-forming diets) ammonium chloride (2.1% , acid forming diet), or potassium chloride (3%, neutral diet, providing K+ and Cl- in amounts equimolar to those in the 4% potassium hydrogencarbonate and the 2.1% ammonium chloride diet, respectively).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: SPF CpB:WU (Wistar random)
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands
- Age at study initiation:  about 5 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: in 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, Institute's cereal-based open formula diet
- Water: ad libitum, tap water
- Acclimation period: no data

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

Details on oral exposure:

DIET PREPARATION
- Institute's cereal-based open formula diet was supplemented with test items
- no further data on diet preparation

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

130 weeks (30 months)

Frequency of treatment:

daily

Dose / conc.:

2 other: % in diet

Dose / conc.:

4 other: % in diet

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Study groups
- control group: unsupplemented institutes cereal based rodent diet
- 2% potassium hydrogencarbonate in diet (approximately 1285 mg/kg bw/d for males and 1576 mg/kg bw/d for females)
- 4% potassium hydrogencarbonate in diet (approximately 2667 mg/kg bw/d for males and 3330 mg/kg bw/d for females)
- Satellite groups: 2.1% NH4Cl (acid-forming diet) and neutral diet supplemented with 3% KCl providing K+ and Cl- in amounts equimolar to those in the 4% KHCO3 diet and the 2.1% NH4Cl diet, respectively
Post-exposure period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, including evaluation regarding grossly visible or palpable masses at regular intervals

BODY WEIGHT: Yes
- Time schedule for examinations: weekly or (from 3 months) once every 4 weeks

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, measured weekly or (from 3 months) once every 4 weeks per cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, calculated per cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes,
- Time schedule for examinations: daily per cage in weeks 1, 4, 6, 8, 12, 55, and 75 (24 hours consumption)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4, 13, 32, 55, and 75 (tail tip blood)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 per group and sex
- Parameters examined: haemoglobin concentration, packed cell volume, red blood cells, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thrombocytes, total white blood cells, prothrombin time, and differential white blood cells

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 5, 14, 32, 56, 76, and 121 (for blood gas analysis)
- Animals fasted: No data
- How many animals: 10 per group and sex
- Parameters examined.:
--Blood gas analysis (tail tip blood): determination of pH, pCO2 and pO2 directly after sampling in capillary tubes, and calculation of bicarbonate and base excess
-- Clinical chemistry: not performed

URINALYSIS: Yes (Urinary acid indices)
- Time schedule for collection of urine: weeks 1, 4, 6, 10, 14, 36, 57, 76, and 129
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food and water for 2-3 h at the start of the light period)
- How many animals: all
- Parameters examined in fasted animals: urinary pH was measured in individual samples, mean pH values were calculated as the negative logarithm of the mean of the hydrogen concentrations instead of as the arithmetical mean of the individual pH values; bicarbonate, titratable acid, ammonia, and urea were measured in pooled samples (two samples per group and sex) and related to creatinine

URINALYSIS: Yes (standard urine parameter)
- Time schedule for collection of urine: weeks 6, 16, 38, and 78
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: 10 per group and sex
- Parameters examined: volume (calibrated tubes), density, gamma glutamyl transferase, creatinine, urea, calcium, potassium, sodium, phosphate, and sulphate

URINALYSIS: Yes (hydroxyprolin)
- Time schedule for collection of urine: weeks 11 and 76
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: 10 per group and sex (two samples from five rats per group and sex)
- Parameter examined: hydroxyproline

URINALYSIS: Yes (Concentrating ability of the kidneys)
- Time schedule for collection of urine: weeks 4, 7, 13, 14, 36, 57, 77, and 129
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, urine collected during the last 16 h of a 24-h water deprivation period, during the 16-h collection period no feed was available
- Parameters examined: volume (graduated tubes), density, and 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, 129

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- CALCIUM CONTENT in FEMUR: month 30 at autopsy, the right femur from all surviving animals was removed, cleaned from adherent soft tissue and weighed, dried at 100 °C, subjected to fat extraction (petroleum ether), redryed and the weight recorded as fat free solid, the fat free femur was ashed at 550 °C and the calcium content was determined by atomic absorption spectrophotometry
- CALCIUM and PHOSPHORUS EXCRETION in URINE and FAECES: In week 53, six rats per sex and 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- week 122 (males) and week 131 (females), all rats were killed on a number of successive working days by exsanguination from the abdominal aorta, under light ether anaesthesia, and subjected to a thorough post-mortem examination. Any abnormalities were recorded and the adrenals, brain, pituitary, kidneys, liver, spleen, testes, ovaries, thyroid, and heart were weight. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
- The following tissues were preserved in 10% neutral phosphate buffered formalin: adrenals, brain, caecum, coagulating gland, colon, epididymides, exorbital lachrimal gland, heart, joint (knee), kidneys, liver, lungs, mammary gland, mesenteric lymph nodes, oesophagus, ovaries, pancreas, parathyroids, parotic salivary glands, pituitary, preputial/clitoral glands, prostate, rectum, seminal vesicles, small intestine (duodenum, ileum, jejunum), spleen, stomach, submaxillary salivary glands, sublingual salivary glands, testes, thymus, thyroid, urinary bladder, uterus, Zymbal glands, and all gross lesions. The lungs and urinary bladder were inflated with the fixative. Paraffin-embedded, 5 µm sections of the above tissues from all animals of each sex in the control group, the potassium hydrocarbon and the ammonium chloride high-dose group were stained with haematoxylin and eosin and examined microscopically. Adrenals, heart, kidneys, stomach, thyroid, testes, urinary bladder, and uterus were also examined in the intermediate-dose groups.

Other examinations:

no

Statistics:

Numerical data were evaluated for statistical significance using one-way analysis of (co)variance [AN(CO)OVA] followed by Dunnett's test, 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

Description (incidence and severity):

- Clinical signs: no treatment-related abnormalities in condition or behaviour; there was only the usual random incidence of ageing symptoms that occur in this strain of rats when maintained over a period of time
- Grossly visible or palpable masses: no treatment-related effects

Mortality:

no mortality observed

Description (incidence):

- Mortality and time of death: mortality rate was not affected by treatment; males were killed in week 122 because mortality in the low-dose group reached 70%,; females were killed after completion of this study in week 131; overall mortality rates at termination in control, low- and high-dose group were 62, 70, and 56% for males, respectively, and 69, 65, and 72% for females, respectively

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- mean body weights decreased in males and females of the high dose group (decrease in the range of 4 to 10%, no exact data given); body weights were also statistically significantly decreased in females of the low dose group at various stages of the study (no further data given)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Food consumption: no treatment-related effects
- ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX:
-- males: 1285 and 2667 mg/kg bw/d (9.3 and 19.3 mmol/kg bw/d)
-- females: 1576 and 3331 mg/kg bw/d (11.4 and 24.1 mmol/kg bw/d)
(recalculation of dose from data given in mmol/kg bw/d (molecular weight=138.21 mg/mmol) )

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

- in the 2% and the 4% dose group approximately 10% and 40% increased, respectively

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- no consistent or treatment-related effects on red blood cell variables, clotting potential or total and differential white blood cell counts

Clinical biochemistry findings:

not examined

Description (incidence and severity):

- Blood gas analysis: dose-related increase in base excess, associated with higher blood pH and bicarbonate concentrations
- Clinical biochemistry: not performed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Urine parameter of non-fasted animals: urine volume increased in both sexes of the high dose group, 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 the high dose group; potassium excretion increased in both sexes in both dose groups; urinary sodium excretion tended to be relatively high at various occasions in rats fed 4% potassium hydrogencarbonate, which may be ascribed to the natriuretic effect of high potassium intake but the figures showed large variations; no treatment related effects on excretion of calcium, phosphate, sulphate, gamma glutamyl transferase or hydroxyproline
- Urinary pH and acid indices: pH increased in both dose groups, net acid excretion (= ammonium ion (NH4+) excretion + urinary titratable acidity - bicarbonate excretion) considerable decreased in both sexes of the high dose group, and to a lesser extend in the low dose group
- Concentrating ability of the kidneys: the renal concentration test (no food and water available prior to and during urine sampling) showed a slightly increased volume associated with a somewhat decreased density of the urine in males of both dose groups and in females of the high dose group in week 77; brownish discoloration of the urine and haematuria, as detected with urinary test stripes and by microscopic examination of the urinary sediment were occasionally increased, but there were no consistent or dose-related differences in incidence or severity of haematuria among the groups; the occurrence of crystals was not affected in any group
- Calcium content in femur: no treatment-related effects

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Kidney weights: relative kidney weights in females tendentiously but neither dose related nor statistically significant increased, relative kidney weights in males tendentiously and dose related but not statistically significant decreased
- Further examined organ weights: there were no consistent or treatment-related changes in the weights of adrenals, brain, testes, liver, spleen, ovaries, pituitary, thyroid, or heart

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Macroscopic examination of the urine bladder: thickening and/or induration of the urinary bladder wall, irregular serosal surface and/or luminal dilatation. Tumorous enlargement of the bladder was seen in one female animal of the 4% ; the mass filled the bladder lumen and was confined to the bladder, i.e. it did not show invasion into, or adhesions with, adjacent tissues. Bladder stones were not observed.
- Macroscopic examination of further organs and tissues: no significant differences among the treatment groups and the controls

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Adrenals: in both sexes of both dose groups, the zona glomerosa was distinctly wider than in controls, the cells in this area were enlarged and showed a finely vacuolar cytoplasm (47-50 animals per sex and dose examined (both survivors and intercurrent deaths), findings in the low and high dose group in 22 (significant) and 41 males (significant, control: 4), respectively, and 8 (not significant) and 32 females (significant, control: 7), respectively. This microscopic finding was interpreted by the study authors as adaptive response to chronic simulation of the adrenal cortex by potassium cations.
- Kidneys: in both sexes of both dose groups, oncocytic tubules but no treatment-related significant severe or very severe nephrosis or urothelial hyperplasia in the pelvis (47-50 animals per sex and dose examined (both survivors and intercurrent deaths), findings in the low and high dose group in 32 (significant) and 36 males (significant, control: 18), respectively, and 22 (significant) and 22 females (significant, control: 1), respectively. The oncocytic tubules were characterised by tubules lined with, often hypertrophy, epithelial cells containing eosinophilic granular cytoplasm (oncocytes), often showing a cystically dilated lumen with epithelial cells protruding into the lumen. As discussed by the study authors, oncocytic tubules do occur spontaneously in untreated rat and are commonly observed in aged males, regarded as regenerative hyperplasia or as a functional tubular hyperplasia in order to meet increased work load. The authors suggested that the hydrogen carbonate anion mainly determined the increased occurrence of this lesion.
- Urinary bladder: in both sexes of both dose groups, the incidence of simple epithelial hyperplasia, of papillary and nodular hyperplasia, and of papillomas were increased. However, except of simple hyperplasia , significance was only reach in high dose female group.
-- Cystitis: in 1/48, 0/50 and 0/49 males, and 1/48, 0/48 and 1/47 (not significant) females in the control, the low- and the high-dose group, respectively
-- Simple epithelial hyperplasia in 1/48, 12/50 (significant) and 22/49 (significant) males, and 1/48, 23/48 (significant) and 24/47 (significant) females in the control, the low- and the high-dose group, respectively
-- Papillary epithelial hyperplasia in 0/48, 0/50 and 0/49 males, and 0/48, 2/48 (not significant) and 5/47 (significant) females in the control, the low- and the high-dose group, respectively
-- Nodular epithelial hyperplasia in 0/48, 2/50 (not significant) and 4/49 (not significant) males, and 0/48, 1/48 (not significant) and 11/47 (significant) females in the control, the low- and the high-dose group, respectively
- Reproductive organs: no treatment-related effects (coagulating gland, epididymides, prostate, seminal vesicles, testes, mammary gland, ovaries, uterus, and preputial/clitoral glands examined)
- Further examined organs and tissues: no treatment-related effects

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Urinary bladder:
--Transitional-cell papilloma (one or multiple) in 0/48, 4/50 (not significant) and 2/49 (not significant) males, and 0/48, 1/48 (not significant) and 6/47 (significant) females in the control, the low- and the high-dose group, respectively
--Transitional-cell carcinoma in 0/48, 1/50 (not significant) and 1/49 (not significant) males, and 0/48, 1/48 (not significant) and 3/47 (not significant) females in the control, the low- and the high-dose group, respectively
- Total tumour incidence: Apart from preneoplastic and neoplastic lesions in the urinary bladder, there were no treatment-related changes in any specific tumor type among the groups; potassium hydrogencarbonate did neither affect type, incidence and multiplicity of tumours, nor time of tumor appearance and the ratio benign-malignant tumours.

HISTORICAL CONTROL DATA
- Neoplastic alterations of urinary bladder: Historical control data obtained in 18 different chronic (exact duration not mentioned) studies with Wistar rats of the laboratory revealed no papillomas or carcinomas of the urinary bladder in 795 male and 777 female control rats.

Other effects:

no effects observed

Description (incidence and severity):

- Calcium content in femur: no treatment-related effects
- Excretion of calcium and phosphorus in faeces and urine: no treatment-related effects

Dose descriptor:

NOAEL

Effect level:

2 667 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

3 331 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

In the urinary bladder the incidence of simple epithelial hyperplasia, of papillary hyperplasia nodular hyperplasia, transitional-cell papilloma were increased. However in the low dose group significant findings were limited to simple epithelial hyperplasia in both sexes. In the high dose group the females showed significantly increased simple, papilary and nodular epithelial hyperplasia and transitional-cell papilloma, but in the males of the high dose group papilary epithelial hyperplasia was missing and nodular epithelial hyperplasia as well as transitional-cell papilloma were not significantly increased. These findings are consistent with a known nongenotoxic mechanism of bladder carcinogenesis in the rat.

The relevance to humans of preneoplastic or neoplastic urinary bladder findings in rats induced by high dose alkali intakes (e.g. as counterions in food additives like artificial sweetener or flavour enhancer) and associated by alkalinization of the urine, elevated urine volumes, altered urine electrolytes composition with or without renal pelvic mineralization or urine precipitation which leads to a cyctotoxic effect on the bladder epithelium with consequent regenerative proliferation and ultimately tumors have been discussed for a long time (e.g. by the International Agency for Research on Cancer as part of the World Health Organization (IARC) in the IARC Monography Vol. 73 (1999) and in the IARC Scientific Publication No 147 (1999) or more recently by the European Food Safety Authority (EFSA) in the option on Aspartam (2006) or a review by Cohen, SM (2008)). It is widely accepted that these high dose effects are specific to the rat and are of no relevance to humans.

The results of this 130 -week (30 month)-study have been published together with the results of a 4-week-study, a 13-week-study, and a 78-week(18 month)-study. The 4-week- and the 13-week-study were separate experiments, the 78-week- and the 130-week-study were started simultaneously. It is not always quite clear, whether the reported results e.g. for urine parameters are the results of a specific study or the summed up results of different studies.

Furthermore, is not quite clear, whether specific parameters (e.g. haematological or urine parameters) were measured until completion of the 78-week-study parallel in two separate groups per sex and dose in the 78-week- and the 130-week-study-groups or only in one group per sex and dose used for both studies.

Quoted references:

Cohen, SM: Review - Thresholds in Genotoxicity and Carcinogenicity: Urinary Bladder Carcinogenicity, Genes and Environment, 40(4), 132 -138 (2008)

European Food Safety Authority (EFSA): Opinion of the Scientific Panel on Food Additives, Flavourings, Processing

Aids and Materials in contact with Food (AFC) on a request from the Commission related to a new long-term carcinogenicity study on aspartame Question number EFSA-Q-2005-122 Adopted on 3 May 2006, The EFSA Journal 356, 1-44 (2006)

World Health Organization International Agency for Research on Cancer (IARC): Some Chemicals that Cause Tumours of the Kidney or Urinary Bladder in Rodents, and Some Other Substances, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Vol. 73 (1999)

World Health Organization International Agency for Research on Cancer (IARC): Species Differences in Thyroid, Kidney and Urinary Bladder Carcinogenesis, IARC Scientific Publication, No 147 (1999)

Executive summary:

In a 130 -week (30-month) toxicity study potassium hydrogencarbonate (a.i. >99.5%) was administered to 15 Wistar rats, strain SPF Cpb:WU/sex/dose in the diet at dose levels of 0, 2 and 4% (0, 1285 and 2667 mg/kg bw/d (9.3 and 19.3 mmol/kg bw/d) in males, respectively and 0, 1576 and 3331 mg/kg bw/d (11.4 and 24.1 mmol/kg bw/d) in females, respectively, based on body weight and food consumption.

Object of the study was to examine the effects of died-induced acid-base disturbances. Albeit the study was not performed according to a explicit mentioned international guideline, accomplishment and documentation cover a broad range of the requirements of the usual guidelines for repeated dose toxicity studies. The dose level exceeds the guideline limit dose for repeated dose toxicity studies which is 1000 mg/kg bw/d in the low dose group by almost 30 or 60 % and in the high dose group by almost 170 or 230 % for males and females, respectively.

The rats adapted relatively easily to the feeding of these very high doses and were free of treatment related adverse effects relevant to humans. Most treatment related changes seen are an expression of the physiological adaptation to the very high ion intake and regarded as of no toxicological relevance. In the urinary bladder significant preneoplastic and neoplastic histopathological epitelial alterations have developed, which are common findings in rats after long term high dose alkali intakes.The relevance to humans of preneoplastic or neoplastic urinary bladder findings in rats induced by high dose alkali intakes (e.g. as counterions in food additives like artificial sweetener or flavour enhancer) and associated by alkalinization of the urine, elevated urine volumes, altered urine electrolytes composition with or without renal pelvic mineralization or urine precipitation which leads to a cyctotoxic effect on the bladder epithelium with consequent regenerative proliferation and ultimately tumors have been discussed for a long time (e.g. by the International Agency for Research on Cancer as part of the World Health Organization (IARC) in the IARC Monography Vol. 73 (1999) and in the IARC Scientific Publication No 147 (1999) or more recently by the European Food Safety Authority (EFSA) in the option on Aspartam (2006) or a review by Cohen, SM on Thresholds in Genotoxicity and Carcinogenicty: Urinary Bladder Carcinogenisis (2008)). It is widely accepted that these high dose effects are specific to the rat and are of no relevance to humans.

The NOAEL relevant to humans is the highest dose tested of 4 % in diet, based on body weight and diet intake corresponding to 2667 mg/kg bw/d (19.3 mmol/kg bw/d) in males and 3331 mg/kg bw/d (24.1 mmol/kg bw/d) in females.

The study has been part of a study set comprising of this 130 -week study, a 4 -week study, a 13 -week study, and a 78 -week (18 -month) study. The dose levels in all studies were 2 and 4% in diet. A summarizing discussion of the results of the whole study set is given in the endpoint summary on repeated dose toxicity.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Non-guideline 4-week-study to examine the effects of died-induced acid-base disturbances. Diets were supplemented with high amounts of potassium hydrogencarbonate (2% or 4%, base-forming diets) or ammonium chloride (2% or 4%, acid forming diets).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: SPF CpB:WU (Wistar random)
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands
- Age at study initiation:  about 5 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: in 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, Institute's cereal-based open formula diet
- Water: ad libitum, tap water
- Acclimation period: no data

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

Details on oral exposure:

DIET PREPARATION
- Institute's cereal-based open formula diet was supplemented with test items
- no further date on diet preparation

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Dose / conc.:

2 other: % in diet

Dose / conc.:

4 other: % in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Study groups:
- control group: unsupplemented institutes cereal based rodent diet 
- 2% potassium hydrogencarbonate in diet (approximately 2920 mg/kg  bw/d)
- 4% potassium hydrogencarbonate in diet (approximately 6100 mg/kg  bw/d)
- Satellite groups: 2% and 4% NH4Cl (acid-forming diets)
Post-exposure period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, measured weekly per cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, calculated per cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily per cage in weeks 1 and 4 (24 hours  consumption)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4 (tail tip blood)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined: haemoglobin concentration, packed cell volume, red blood cells, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thrombocytes, total white blood cells, prothrombin time, and differential white blood cells

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 5 (blood gas analysis, clinical chemistry parameter), week 4 (glucose analysis)
- Animals fasted: No data (except on glucose analysis)
- How many animals: all
- Parameters examined.:
-- Blood gas analysis (tail tip blood): determination of pH, pCO2 and pO2 directly after sampling in capillary tubes, and calculation of bicarbonate and base excess
-- Clinical chemistry:
--- Plasma from abdominal aorta collected at necropsy: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, lactate dehydrogenase, total protein, albumin, total bilirubin, urea, creatinine, calcium, inorganic phosphate, chloride, sodium, and potassium
--- Whole blood collected from the tip of the tail in week 4 after overnight fasting: glucose

URINALYSIS: Yes (Urinary acid indices)
- Time schedule for collection of urine: weeks 1 and 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food and water for 2-3 h at the start of the light period)
- How many animals: all
- Parameters examined: urinary pH was measured in individual samples, mean pH values were calculated as the negative logarithm of the mean of the hydrogen concentrations instead of as the arithmetical mean of the individual pH values; bicarbonate, titratable acid, ammonia, and urea were measured in pooled samples (two samples per group and sex) and related to creatinine

URINALYSIS: Yes (Concentrating ability of the kidneys)
- Time schedule for collection of urine: week 4
- Metabolism cages used for collection of urine: No data
- How many animals: all
- Animals fasted: Yes, urine collected during the last 16 h of a 24-h water deprivation period, during the 16-h collection period no feed was available
- Parameters examined: volume (calibrated tubes), density, and in addition, examinations for protein, glucose, occult blood, ketones, bilirubin, and urobilinogen, appearance, and microscopy of the sediment

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:

OTHER:
- CALCIUM CONTENT in FEMUR: week 4 at autopsy, the right femur from 10 animals per group and sex was removed, cleaned from adherent soft tissue and weighed, dried at 100 °C, subjected to fat extraction (petroleum ether), redryed and the weight recorded as fat free solid, the fat free femur was ashed at 550 °C and the calcium content was determined by atomic absorption spectrophotometry

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- end of week 4, all rats were killed on a number of successive working days by exsanguination from the abdominal aorta, under light ether anaesthesia, and subjected to a thorough post-mortem examination. Any abnormalities were recorded and the adrenals, brain, pituitary, kidneys, liver, spleen, testes, ovaries, thyroid, thymus, and heart were weight. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
- The following tissues were preserved in 10% neutral phosphate buffered formalin: adrenals, kidneys, liver, lungs, mesenteric lymph nodes, spleen, testes, urinary bladder, and all gross lesions. The lungs and urinary bladder were inflated with the fixative. Paraffin-embedded, 5 µm sections of the above tissues from all animals of each sex in the control group, the potassium hydrocarbon and the ammonium chloride high-dose group were stained with haematoxylin and eosin and examined microscopically. Adrenals, kidneys, testes, and urinary bladder were also examined in the intermediate-dose groups.

Other examinations:

no

Statistics:

Numerical data were evaluated for statistical significance using one-way analysis of (co)variance [AN(CO)OVA] followed by Dunnett's test, 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

Description (incidence and severity):

no treatment-related abnormalities in condition or behaviour

Mortality:

no mortality observed

Description (incidence):

Mortality and time of death: no animal died

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no treatment-related effects

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Food consumption: no treatment-related effects
- ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX:
-- males: 2944 and 6054 mg/kg bw/d (21.3 and 43.8 mmol/kg bw/d)
-- females: 2902 and 6137 mg/kg bw/d (21.0 and 44.4 mmol/kg bw/d)
(recalculation of dose from data given in mmol/kg bw/d (molecular weight=138.21 mg/mmol) )

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

- in the 2% and the 4% dose group approximately 10% and 40% increased, respectively

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- no consistent or treatment-related effects on red blood cell variables, clotting potential or total and differential white blood cell counts

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Blood gas analysis: dose-related increase in base excess, associated with higher blood pH and bicarbonate concentrations
- Clinical biochemistry: potassium levels in plasma were generally increased in males and females fed 2 or 4% potassium hydrogencarbonate, although the differences with the controls were not always statistically significant; significant base excess in both sexes of both dose groups; no treatment-related effects on further measured blood clinical biochemistry parameters

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Urine parameter of non-fasted animals: not performed
- Urinary pH and acid indices: pH increased in both dose groups (pH values: 8.5 to 8.8 (control: 7.6 to 7.7)), net acid excretion (= ammonium ion (NH4+) excretion + urinary titratable acidity - bicarbonate excretion) considerable decreased in both sexes of the high dose group, and to a lesser extend in the low dose group
- Concentrating ability of the kidneys: the renal concentration test (no food and water available prior to and during urine sampling) showed a slightly increased volume associated with a somewhat decreased density of the urine in males of both dose groups; brownish discoloration of the urine and haematuria, as detected with urinary test stripes and by microscopic examination of the urinary sediment were occasionally increased, but there were no consistent or dose-related differences in incidence or severity of haematuria among the groups; the occurrence of crystals was not affected in any group

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Kidney weight: relative kidney weights in both sexes of both dose groups tendentiously but not statistically significant increased
- Further examined organ weights: there were no consistent or treatment-related changes in the weights of adrenals, brain, testes, liver, spleen, ovaries, pituitary, thyroid, thymus or heart

Gross pathological findings:

no effects observed

Description (incidence and severity):

- macroscopic examination at necropsy did not reveal significant differences among the treatment groups and the controls

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- no significant differences among the groups treated with potassium hydrogencarbonate and the controls; no treatment-related significant effects
- examination of the urinary bladder showed (not significant) very slight to slight simple epithelial hyperplasia in 1/10 and 2/10 males (control: 0/10) and 1/10 and 3/10 females (control: 0/10) in the low and the high dose groups, respectively

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Calcium content in femur: no treatment-related effects

Dose descriptor:

NOAEL

Effect level:

6 054 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Highest dose tested (4 % in diet); no adverse effects relevant to humans observed; only adaptive effects on very high ion ingestion seen.

Dose descriptor:

NOAEL

Effect level:

6 137 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: Highest dose tested (4 % in diet); no adverse effects relevant to humans observed; only adaptive effects on very high ion ingestion seen.

Critical effects observed:

no

The results of this 4-week-study have been published together with the results of a 13-week-study, a 78-week(18 month)-study, and a 130-week (30 month)-study. The 4-week- and the 13-week-study were separate experiments, the 78-week- and the 130-week-study were started simultaneously. It is not always quite clear, whether the reported results e.g. for urine parameters are the results of a specific study or the summed up results of different studies.

Executive summary:

In a 4 -week toxicity study potassium hydrogencarbonate (a.i. >99.5%) was administered to 10 Wistar rats, strain SPF Cpb:WU/sex/dose in the diet at dose levels of 0, 2 and 4% (0, 2944 and 6054 mg/kg bw/d (0, 21.3 and 43.8 mmol/kg bw/d) in males, respectively and 0, 2902 and 6137 mg/kg bw/d (0, 21.0 and 44.4 mmol/kg bw/d) in females, respectively, based on body weight and food consumption.

Object of the study was to examine the effects of died-induced acid-base disturbances. Albeit the study was not performed according to a explicit mentioned international guideline, accomplishment and documentation cover a broad range of the requirements of the usual guidelines for repeated dose toxicity studies. The dose level exceeds the guideline limit dose for repeated dose toxicity studies which is 1000 mg/kg bw/d by a factor of almost 3 and of more than 6 in the low and high dose group, respectively.

The rats adapted relatively easily to the feeding of these very high doses and were free of treatment related adverse effects. The few treatment related changes seen are mostly an expression of the physiological adaptation to the very high ion intake and regarded as of no toxicological relevance.

The NOAEL is the highest dose tested of 4 % in diet, based on body weight and diet intake corresponding to 6054 mg/kg bw/d (43.8 mmol/kg bw/d) in males and  6137 mg/kg bw/d (44.4 mmol/kg bw/d) in females.

The study has been part of a study set comprising of this 4-week study, a 13-week study, a 18-month, and a 30-month study. The dose levels in all studies were 2 and 4% in diet. A summarizing discussion of the results of the whole study set is given in the endpoint summary on repeated dose toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

Wistar rats were maintained on diets containing 2 or 4% of of potassium hydrogencarbonate over 4-weeks, 13-weeks, 18-months, and 30-months (Lina 2004, Lina and Kuijpers 2004, Lina, Hollanders and Kuijpers 1994). Object of the studies was to examine the effects of died-induced acid-base disturbances. The studies were not performed according to an explicitly mentioned international guideline, but accomplishment and documentation cover a broad range of the requirements of the usual guidelines for repeated dose toxicity studies. Thus, the studies are relevant, reliable and adequate for the purpose of assessing repeated dose toxicity. Even though the rats were treated with very high doses of potassium hydrogencarbonate in their feed, in the highest dose groups corresponding to daily intakes of more than or nearly 3000 mg/kg bw in the 18- and 30-months studies to even more than 6000 mg/kg bw in the 4-week study, and in the lowest dose groups of still distinctly more than the guideline limit dose of repeated dose toxicity studies (1000 mg/kg bw) in the 30-months study to approximately 2900 mg/kg bw in the 4-weeks study, only few and mainly adaptive responses to the treatment were seen. Animals which received 18 months or longer 4% potassium hydrogencarbonate in their feed showed a slightly impaired body weight. In the blood, increased potassium levels and base excess, associated with higher blood pH and bicarbonate concentrations were seen. The water intake was increased up to 40% and also the urinary volume, potassium excretion and pH were distinctly increased, the urinary net acid excretion was decreased. The microscopic alterations seen in the adrenals and the kidneys may be interpreted as adaptive response to chronic simulation of the adrenal cortex by potassium cations and as a functional adaptation in order to compensate the increased work load of the kidneys, respectively and regarded as of no toxicological relevance. The missing relevance to humans of preneoplastic and neoplastic histopathological epitelial alterations in the urinary bladder are discussed in Chapter 5.8 Carcinogenicity. Thus, up to and including the highest dose tested of 4 % potassium hydrogencarbonate in the diet administered for 4 weeks, 13 weeks, 18 months or almost a lifetime (30 months), the rats adapted relatively easily to the feeding of these very high dose levels far exceeding the guideline limit dose of repeated dose toxicity studies and were free of treatment related adverse effects relevant to humans.

The NOAELs of potassium hydrogencarbonate relevant to humans is the highest dose tested of 4 % in diet, based on body weight and diet intake correspond to

- 6054 mg/kg bw/d (43.8 mmol/kg bw/d) in males and 6137 mg/kg bw/d (44.4 mmol/kg bw/d) in females in the 4-weeks-study

- 4326 mg/kg bw/d (31.3 mmol/kg bw/d) in males and 4879 mg/kg bw/d (35.3 mmol/kg bw/d) in females in the 13-weeks-study

- 2861 mg/kg bw/d (20.7 mmol/kg bw/d) in males and 3566 mg/kg bw/d (25.8 mmol/kg bw/d) in females in the 18-months study and of

- 2667 mg/kg bw/d (19.3 mmol/kg bw/d) in males and 3331 mg/kg bw/d (24.1 mmol/kg bw/d) in females in the 30-months study

Based on these study results, the approved use of potassium hydrogencarbonate as well as potassium carbonate in pharmaceutical preparations and foodstuffs with no specific quantity restriction except of the quantum satis principle and the nutritional essentiality of potassium as well as the essential role of carbonate in the body, potassium hydrogencarbonate can be judged as systemically non-toxic. Thus, there is no need to set DNELs for systemic repeated dose toxicity.

Justification for classification or non-classification

There is no evidence for intrinsic toxic properties relevant to humans obtained from the results of reliable and adequate subacute, subchronic and chronic oral studies on rats. The tested dose range far exceeds the guideline limit dose of repeated dose toxicity studies.

Therefore, no classification is required for repeated dose toxicity according to CLP Regulation (EC) No 1272/2008 with respect to systemic toxicity.