Potassium carbonate

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

The role of alkalizing and neutral potassium salts in urinary bladder carcinogenesis in rats.

Author:

Lina BAR, Hollanders VMH, Kuijpers MHM

Year:

1994

Bibliographic source:

Carcinogenesis 15: 523-527

Reference 2

Reference Type:

publication

Title:

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

Author:

Lina BAR, Kuijpers MHM

Year:

2004

Bibliographic source:

Food Chem. Toxicol. 42: 135-153

Reference 3

Reference Type:

other: dissertation thesis

Title:

Diet-induced modification of the acid-base balance in rats - Toxicological implications, Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht, The Netherlands

Author:

Lina BAR

Year:

2004

Bibliographic source:

ISBN 90-393-3843-4 Printed by Febodruk BV, Enschede, The Netherlands, available via Internet: http://igitur-archive.library.uu.nl/dissertations/2004-1126-100141/full.pdf

Materials and methods

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

Test material

Test material information

Constituent 1

Reference substance name:

Potassium hydrogencarbonate

EC Number:

206-059-0

EC Name:

Potassium hydrogencarbonate

Cas Number:

298-14-6

Molecular formula:

CH2O3.K

IUPAC Name:

potassium hydrogen carbonate

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 other: % in diet

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.