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Repeated dose toxicity: oral

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Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and reproduction toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Very well-documented study which allows derivation of a NOAEL value for chronic toxicity. Most reliable study for NOAEL deriviation for repeated dose toxicity effects of sodium metabisulfite.
Justification for type of information:
see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Very well-documented study which allows derivation of a NOAEL value for chronic toxicity. Most reliable study for NOAEL deriviation for repeated dose toxicity effects of sodium metabisulfite.
Justification for type of information:
see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a three-generation feeding study, groups of 20 male and 20 female Wistar rats received 0, 0.125, 0.25, 0.5, 1.0 and 2.0% sodium metabisulphite, i.e. 49, 108, 220, 460, and 955 mg/kg bw/d as actual dose in a thiamine-containing diet over periods of 2 years.
GLP compliance:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: derived from the Institute's Wistar-derived colony
- Age at study initiation: weanling or young rats.
- Housing: housed in groups of 5 in screen-bottomed cages
- Diet: ad libitum, basal diet was Institute's stock diet
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24-26
No further details are given.
Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): diets were freshly prepared every 2 weeks.
- Sulphite was added by mechanical mixing of Na2S2O5 at levels varying from 0.125 to 8%. The basal diet was Institute's stock diet, with following percentage composition: 35% ground yellow maize, 26& ground whole wheat, 10% soyabean-oil meal, 8% fish meal, 4% meat scraps, 2.7% dried whey, 3% brewer's yeast, 3.3% vitamin preparations, 1.5% minerals, 0.5% NaCl, 3% soyabean-oil, 3% grass meal.
- In order to compensate for the destruction of thiamine by sulphite, the stock diet was drastically enriched with 50ppm thiamine.
- Storage temperature of food: diets were stored at -18°C and the rats were provided daily with a fresh portion of previously frozen diet.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diets were analysed for SO2 and thiamine.
Duration of treatment / exposure:
Exposure period: 104 weeks (F0 and F1 generation) and 30 weeks (F2 generation)
Premating exposure period (males): 21 weeks
Premating exposure period (females): 21 weeks
Duration of test: until the weaning of the F3 animals
Frequency of treatment:
continuously (in diet)
Post exposure period:
no data
Remarks:
Doses / Concentrations:
0.125 % (ca. 50 mg/kg bw)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.25 % (ca. 110 mg/kg bw)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.5 % (ca. 220 mg/kg bw)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.0 % (ca. 460 mg/kg bw)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2.0% (ca. 960 mg/kg bw)
Basis:
nominal in diet
No. of animals per sex per dose:
20 rats per sex and group
Control animals:
yes, plain diet
Details on study design:
no data
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at week 32, 64, and 100 all rats of the F0 and F1a generation and at week 28 those of the F2a generations were examined for occult blood in faeces.

BODY WEIGHT: Yes
- Time schedule for examinations: in all generations, changes of body weight were recorded weekly for the first 12 weeks and once every four week thereafter.

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
- Time schedule for examinations: food consumption of each diet group was measured at intervals during 1-week periods.

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes.
- Time schedule for collection of blood/How many animals: in F0 generation at week 52, 78, and 100; at week 52 and 102 in the F1a generation, and at week 20 of F2 generation.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters checked : haemoglobin concentration, haematocrit value, numbers of erythrocytes, total and individual types of leukocytes were counted.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at week 52 and 104.
- Animals fasted: No data
- How many animals: in F0 generation rats.
- Parameters checked: glutamic-oxalacetic and glutamic-pyruvic transaminases.

URINALYSIS: Yes
- Time schedule for collection of urine: at week 13, 28, 52, 78, and 101 in F0; at week 28, 52 and 100 in F1 and at week 28 in the F2.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- How many animals: 5-11 rats of each sex.
- Parameters checked: concurrently pooled urine analysis for appearance, pH, glucose, albumin, occult blood, ketones, and microscopy of the sediment.

NEUROBEHAVIOURAL EXAMINATION: No

No further information given.
Sacrifice and pathology:
SACRIFICE: Yes
- After 52 weeks, 5 males and 5 females of each diet group in the F0 generation were killed for interim organ weights analysis and gross pathology.
- At week 104 all survivors of the F0 and F1-generations were killed.
- At about week 30 all survivors of the F2 generation were killed.

GROSS PATHOLOGY: Yes,
- At week 104 all survivors in the F0 and F1 generations and at about 30 weeks those of the F2 generation were killed and autopsied.
- Rats that died or killed when moribund were also autopsied, but tissue samples were preserved only if autolysis was not too advanced.
- The heart, kidneys, liver, spleen, brain, testes, ovaries, pituitary, thyroid and adrenals were weighed.

HISTOPATHOLOGY: Yes
- Tissue samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm and stained with haematoxylin and eosin.
- The following tissues were examined: heart, kidneys, liver, spleen, brain, testes, ovaries, pituitary, thyroids, parathyroids, adrenals, thymus, lungs, trachea, salivary glands, gastro-intestinal tract, pancreas, urinary bladder, skeletal muscle, spinal cord, femoral nerve, skin, bone marrow, axillary and mesenteric lymph nodes, exorbital lachrymal gland, aorta, mammary glands, uterus, prostate, seminal vesicle and coagulating gland.
Other examinations:
- Thiamine was determined in the pooled urine samples of 5-10 male and 5 - 10 female rats of each group in the three generations at several stages and also in pooled liver samples of five F0-generation rats of each sex at week 52 and 104.

Statistics:
no data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
occult blood in faeces
Mortality:
mortality observed, treatment-related
Description (incidence):
occult blood in faeces
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
no dose related effects
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no apparent effect
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
changes in gastric morphology
Histopathological findings: neoplastic:
no effects observed
Details on results:
DIETARY LEVELS
- The losses were 22, 14, 12, 8 and 4.5% Na2S2O5 and 2.7, 1.7, 8.3, 14.5 and 15.4 % thiamine in the diets supplemented with 0.125, 0.25, 0.5, 1 and 2 % sulfite, respectively.

CLINICAL SIGNS AND MORTALITY
- The general conditions of the rats remained good during the first 72 weeks in the F0 generation as well as in the two descendent generations.
- After this time, aging symptoms developed in many rats and mortality increased rapidly in nearly all groups.
- The survival in the sulphite groups was generally higher than in the controls, except in F1 males with 2.0% sulphite.
- No deaths occurred in the females of the same group.
- Occult blood was present in the faeces of all generations at the highest dose level of 2.0%, and in only 13-60% of the animals on the 1% sulphite diet.

BODY WEIGHT AND WEIGHT GAIN
- There was a marginal reduction in body weight gain in both sexes of the F1 and F2 generations given 2% metabisulphite.
- Some effects (without dose relation) were present in F1 females given 0.125, 0.25 and 0.5% sulphite and those given 0.25 and 0.5% in the F2 generation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- There were no distinct differences in food consumption.

HAEMATOLOGY
- Haemoglobin, haematocrit values and erythrocyte counts were marginally reduced in F0 females at 2% metabisulphite.

CLINICAL CHEMISTRY
- Significant decreases in serum glutamic-pyruvic-transaminase values occured at wk 104 in male rats of the F0-generation receiving 0.125 % sulfite.
- Otherwise, there were no differences between the test and control animals of this generation in transaminase activities

URINALYSIS
- Kidney function: phenol-red excretion, specific gravity and glutamic-oxalacetic-transaminase activity in the urine were not adversely affected.
- Urine analysis values were essentially normal-

ORGAN WEIGHTS
- Interim results obtained after 1 year did not indicate any effect of sulfite on organ to body weight ratios.
- Terminally the relative weights of the livers of the F0- and F1-generation rats were lower in all the test groups than in the controls, but there was no evidence of a dose-related response; in the F2-generation, no distinct decrease in the liver weigth was observed.
- Relative weights of the kidneys were increased by the 2% sulphite level in the F2 females only, but this increase was accompanied by neither functional nor histological changes.

GROSS PATHOLOGY
- Pathological changes attributable to the feeding of sulphite were observed only in the stomach.
- A distinctly raised and thickened limiting ridge and small amounts of a reddish-brown flocky material in the mucus layer of teh glandular stomach were seen grossly in the groups given the two highest sulphite levels.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Hyperplastic changes were seen in both the forestomach and glandular stomach at the 1.0 and 2.0% sulphite level in each of the three generations.
- At the 0.5% level, treatment-related lesions were seen only in a few male and female animals of the F2 generation.
- The gastric changes were treatment related.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- There was no indication that metabisulphite had any carcinogenic effect.
- The number of lymphoreticular pulmonary tumours in males decreased with increasing levels of sulphite in the diet.
- The incidence of thyroid and pituitary tumours in the control group of the males was exceptionally low, whereas those noted in the various test groups represented numbers normally found in the strain used-
- All other neoplasms occurred in a random manner with no apparent relationsship between number, location of tumours and treatment.

OTHER FINDINGS
- The 2% sulphite group showed no distinct changes in the thiamine status indicating prevention of thiamine deficiency even at a dietary level of 2% metabisulphite.
Relevance of carcinogenic effects / potential:
There was no indication that metabisulphite had any carcinogenic effect.
The study described is not a conventional carcinogenicity study as the animals were mated to determine reproductive performance. Nevertheless, this data is sufficient to assess the carcinogenic potential of Na2S2O5 since the animals were maintained for 104 weeks and suitable histological examinations were performed.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
108 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
> 955 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No signs of systemic toxicity were observed and the NOAEL can be expected above the highest dose of 2% metabisulphite corresponding to 955 mg/kg bw/d of Na2S2O5 (or 640 mg/kg bw/d as SO2 equivalents).
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
> 955 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no indication that metabisulphite had any carcinogenic effect at the highest dietary dose of 2% Na2S2O5 (955 mg/kg bw/d or 640 mg/kg bw/d as SO2 equivalents).
Remarks on result:
not determinable
Remarks:
no NOAEL identified. Effect type:carcinogenicity (migrated information)

Calculation of the dose level in mg/kg bw/d was done by converting % diet into ppm and by assuming an average body weight for older rats of 400 g and a daily feed intake of 20 g as follows:

0.215 % = 2150 ppm x 0.05 = 108 mg/kg bw/d Na2S2O5, equivalent to 72 mg/kg bw/d SO2

1.91% = 19100 ppm x 0.05 = 955 mg/kg bw/dNa2S2O5, equivalent to 640 mg/kg bw/d SO2

Conclusions:
No evidence of a carcinogenic activity of sodium metabisulphite was seen in a three-generation feeding study at dietary dose up to 2% Na2S2O5 (955 mg/kg bw/d or 640 mg/kg bw/d as SO2 equivalents).
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study on gastric lesions in rats induced by Na2S2O5.
Justification for type of information:
see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In short-term studies on male and female Wistar rats (10 to 56 days) sodium metabisulphite was added to the diet at levels of 0, 0.5, 1, 2, 4, 6 and 8%; in long-term studies (8, 12 and 24 months) at levels of 0, 0.125, 0.25, 0.5, 1 and 2%. Pathological and microscopic examinations of the stomach of the rats were performed after treatment periods of 10, 28 and 56 days (short-term) and after 8, 12 and 24 months.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: from Institute's colony (Central institute for Nutrition and Food Research)
- Diet: ad libitum
- Water: tap water, ad libitum
Route of administration:
oral: feed
Vehicle:
other: diet
Details on oral exposure:
DIET PREPARATION
- Sodium metabisulphite was added to the diet at levels of 0, 0.5, 1, 2, 4, 6 and 8% in the short-term studies.
- Sodium metabisulphite was added to the diet at levels of 0, 0.125, 0.25, 0.5, 1 and 2% in the long-term studies.
- Thiamine deficiency was prevented by addition of 50 mg thiamine/kg food.
- Diets were stored at -18 °C and the animals were daily provided with a fresh portion of frozen diet.
Duration of treatment / exposure:
Short-term studies: 10 to 56 days
Long-term studies: 8, 12, and 24 months
Frequency of treatment:
continuously (in diet)
Remarks:
Doses / Concentrations:
0.125 % (long-term study)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.25 % (long-term study)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.5 % (long-term study)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.0 % (short- and long-term study)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2.0 % (short- and long-term study)
Basis:
nominal in diet
No. of animals per sex per dose:
no data
Control animals:
yes, plain diet
Details on study design:
Doses of short-term study were: 0, 0.5, 1, 2, 4, 6 and 8 % sodium metabisulphite.
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite

DETAILED CLINICAL OBSERVATIONS: Yes, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite

BODY WEIGHT: Yes, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite

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: Yes, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite

CLINICAL CHEMISTRY: Yes, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite

URINALYSIS: Yes, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes
- Pathological and microscopic examinations of the stomach of the rats were performed after treatment periods of 10, 28 and 56 days (short-term) and after 8, 12 and 24 months.
- Rats were killed by decapitation and the stomach was removed, examined grossly and fixed in a 4 % aqueous neutral phosphate-buffered solution of formaldehyde. After embedding in paraffin, 5 µm thick sections were stained and examined microscopically. Selected sections were stained with periodic acid-Schiff (PAS) and Alcian blue.
Other examinations:
no
Statistics:
no data
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at dietary levels of 0.5 % and higher
Histopathological findings: neoplastic:
not specified
Details on results:
- In general, the lesions observed in the long-term experiments were not more pronounced than in the short-term tests at corresponding levels.
- For results of other examinations, see section 7.5.1 Repeated dose toxicity:oral: K_Til_1972_sodium metasulfite.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Feeding of metabisulphite induced hyperplastic and inflammatory changes in the forestomach at dietary levels of 0.5 % and higher.
- The hyperplasia mainly consisted of hyperkeratosis, acanthosis and papillomatous elevations; the inflammatory changes comprised ulcerations and mild cellular infiltrates in the submucosa.
- The metabisulphite lesions induced in the glandular stomach consisted of haemorrhagic micro-erosions, necrosis of epithelial cells, cellular infiltrations, and an atypical glandular hyperplasia.
- A mild atrophic gastritis developed in about 30% of the rats treated with 2% metabisulphite for 2 years.
- Neither the results of the short-term tests nor those of the long-term experiments gave any evidence of metabisulphite feeding damaging the pyloric part of the stomach.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- The microscopic examinations gave no evidence for the formation of tumours in the stomach.
Dose descriptor:
NOAEL
Remarks:
local toxicity
Effect level:
108 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Calculation of the dose level in mg/kg bw/d was done by converting % diet into ppm and by assuming an average body weight for older rats of 400 g and a daily feed intake of 20 g as follows:

0.215 % = 2150 ppm x 0.05 = 108 mg/kg bw/d Na2S2O5, equivalent to 72 mg/kg bw/d SO2

1.91 % : 19100 ppm x 0.05 = 955mg/kg bw/d Na2S2O5, equivalent to 640 mg/kg bw/d SO2

Conclusions:
Chronic treament of rats at higher dietary levels reulted in the occurance of blood in faeces and hyperplastic and inflammatory changes in the forestomach mucosa. Based on these findings, a sodium metabisulphite level of 0.25% can be considered as a NOAEL for local effects corresponding to 108 mg/kg bw/d Na2S2O5 (or about 72 mg/kg bw/d as SO2 equivalents).

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
The Toxicity of Sulphite. I. Long-term feeding and multigeneration studies in rats.
Author:
Til, H.P., et al.
Year:
1972
Bibliographic source:
Food and Cosmetics Toxicology, 10: 291-310.
Reference Type:
review article or handbook
Title:
SIDS Dossier on Sodium Disulphite
Author:
Anonymous
Year:
2001
Bibliographic source:
Final Draft for Publication, available on http://www.oecd.org/document/63/0,2340,en_2649_34379_1897983_1_1_1_1,00.html

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a three-generation feeding study, groups of 20 male and 20 female Wistar rats received 0, 0.125, 0.25, 0.5, 1.0 and 2.0% sodium metabisulphite, i.e. 49, 108, 220, 460, and 955 mg/kg bw/d as actual dose in a thiamine-containing diet over periods of 2 years.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium disulphite
EC Number:
231-673-0
EC Name:
Disodium disulphite
Cas Number:
7681-57-4
Molecular formula:
Na2S2O5
IUPAC Name:
disodium disulphite
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): sodium metabisulfite (obtained from Amsterdamsche Chinine Fabriek (ACF), Amsterdam, Netherlands)
- Molecular formula (if other than submission substance): Na2S2O5
- Analytical purity: between 95 and 99%
Specific details on test material used for the study:
not specified

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: derived from the Institute's Wistar-derived colony
- Age at study initiation: weanling or young rats
- Housing: housed in groups of 5 in screen-bottomed cages
- Diet (ad libitum): basal diet was Institute's stock diet, with following percentage composition: 35% ground yellow maize, 26 % ground whole wheat, 10% soyabean-oil meal, 8% fish meal, 4% meat scraps, 2.7% dried whey, 3% brewer's yeast, 3.3% vitamin preparations, 1.5% minerals, 0.5% NaCl, 3% soyabean-oil, 3% grass meal.
- Water (ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature: 24 - 26 °C

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
In view of the widespread use of sulphite in foods and drinks it was considered desirable to re-examine the toxicity of sulphite administered in the diet.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): diets were freshly prepared every 2 weeks.
- Sulphite was added by mechanical mixing of Na2S2O5 at levels varying from 0.125 to 8%.
- In order to compensate for the destruction of thiamine by sulphite, the stock diet was drastically enriched with 50 ppm thiamine.
- Storage temperature of food: diets were stored at -18°C and the rats were provided daily with a fresh portion of previously frozen diet.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diets were analysed for SO2 and thiamine.

Results:
- SO2 determinations on the diets showed considerable losses of sulphite.
- proportionally the losses of sulphite decreased with increasing dietary levels of sulphite both immediately after mixing and after storage for 2 wk at - 18 °C and subsequent storage for 24 hr at room temperature.
- thiamine content decreased after storage for 2 wk at -18'C only in the 1 and 2% sulphite diets. Subsequent storage for 24 hr at room temperature showed slight and similar losses at 0, 0.125 and 0.25 %, but at higher sulphite levels there was a definite tendency towards greater losses with an increase of sulphite in the diet.
- during the experimental period, the rats consumed food that was stored on average for 1 week at -18°C and subsequently kept in the feeders for 12 hr at 24°C. The losses amounted to 22, 14, 12, 8 and 4.5 % Na2S20s and 2.7, 1.7, 8.3, 14.5 and 15.4% thiamine in the diets supplemented with 0.125, 0.25, 0.5, 1 and 2 % sulphite, respectively.


Duration of treatment / exposure:
104 weeks (21 weeks or 34 weeks before mating and up to a total of 104 weeks)
Frequency of treatment:
continuously
Doses / concentrationsopen allclose all
Dose / conc.:
0.125 other: %
Remarks:
ca. 50 mg/kg bw (nominal in diet)
Dose / conc.:
0.25 other: %
Remarks:
ca. 110 mg/kg bw (nominal in diet)
Dose / conc.:
0.5 other: %
Remarks:
ca. 220 mg/kg bw (nominal in diet)
Dose / conc.:
1 other: %
Remarks:
ca. 460 mg/kg bw (nominal in diet)
Dose / conc.:
2 other: %
Remarks:
ca. 960 mg/kg bw (nominal in diet)
No. of animals per sex per dose:
20 males / 20 females
Control animals:
yes, plain diet
Details on study design:
not specified
Positive control:
not specified

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs and mortality
At week 32, 64 and 100 all rats were examined for occult blood in the faeces.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 12 weeks and once every four week thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations of food consumption: at intervals during 1-week periods.

FOOD EFFICIENCY: Not specified
WATER CONSUMPTION: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 52, 78, and 100
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 6 – 10 animals of each sex from the control, 1 and 2 % groups
- Parameters checked: haemoglobin concentration, haematocrit value, numbers of erythrocytes, total and individual types of leukocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 52 and 104.
- Animals fasted: Not specified
- How many animals: not specified
- Parameters checked: glutamic-oxalacetic transaminases and glutamic-pyruvic transaminases.

URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 28, 52, 78, and 101
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- How many animals: 5-11 rats of each sex, usually from the controls and the 1 and 2 % groups
- Parameters checked: phenol-red excretion. glutamic-oxalacetic transaminases, specific gravity, appearance, pH, glucose, albumin, occult blood, ketones, and microscopy of the sediment.

NEUROBEHAVIOURAL EXAMINATION: Not specified
Sacrifice and pathology:
SACRIFICE: Yes
- After 52 weeks, 5 males and 5 females of each diet group were killed for interim organ weights analysis and gross pathology.
- At week 104 all survivors were killed.

GROSS PATHOLOGY: Yes
- At week 104 all survivors were killed and the heart, kidneys, liver, spleen, brain, testes, ovaries, pituitary, thyroid and adrenals were weighed as well as fixed.
- Rats that died or were killed when moribund were also autopsied, but tissue samples were preserved only if autolysis was not too advanced.

HISTOPATHOLOGY: Yes
- Tissue samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm and stained with haematoxylin and eosin.
- The following tissues were examined: heart, kidneys, liver, spleen, brain (three levels), testes, ovaries, pituitary, thyroids, parathyroids, adrenals, thymus, lungs, trachea, salivary glands, gastro-intestinal tract (six levels), pancreas, urinary bladder, skeletal muscle, spinal cord, femoral nerve, skin, bone marrow (sternum), axillary and mesenteric lymph nodes, exorbital lachrymal gland, aorta, mammary glands, uterus, prostate, seminal vesicle and coagulating gland.
Other examinations:
- Thiamine was determined in the pooled urine samples of 5-10 male and 5 - 10 female rats of each group at several stages and also in pooled liver samples of five rats of each sex at week 52 and 104.
Statistics:
not specified

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- All rats in the highest dose group showed indications of occult blood in the faeces, while this effect occurred in only 21-60% of the animals on diets containing 1 % sulphite.
- In 10 % of the females given 0.25 % and in 10 % of the males given 0.5 % sulphite slight indications of intestinal blood loss were observed at wk 32 only.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Pathological changes attributable to the feeding of sulphite were observed only in the stomach.
- A distinctly raised and thickened limiting ridge and small amounts of a reddish-brown flocky material in the mucus layer of the glandular stomach were seen grossly in the groups given the two highest sulphite levels.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Hyperplastic changes were seen in both the forestomach and glandular stomach at the 1.0 and 2.0% sulphite level. They were either present only at the 1 and 2 % dosage levels or their incidence was distinctly increased at these levels.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- General conditions of the rats remained good during the first 72 weeks.
- After this time, aging symptoms developed in many rats and mortality increased rapidly.
- The survival in the sulphite groups was generally higher than in the controls.

BODY WEIGHT AND WEIGHT GAIN
- Body weights were comparable irrespective of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE
- There were no distinct differences in food consumption.

HAEMATOLOGY
- Haemoglobin, haematocrit values and erythrocyte counts were marginally reduced at 2% sulphite at week 52, 78, and 100.

CLINICAL CHEMISTRY
- Significant (P < 0.05) decreases in serum glutamic-pyruvic-transaminase values occured at wk 104 in male rats receiving 0.125 % sulfite.
- Otherwise, there were no differences between the test and control animals in transaminase activities either at week 52 or at week 104.

URINALYSIS
- Kidney function: phenol-red excretion, specific gravity and glutamic-oxalacetic-transaminase activity in the urine were not adversely affected.
- Urine analysis values were essentially normal.

ORGAN WEIGHTS
- Interim results obtained after 1 year did not indicate any effect of sulphite on organ to body weight ratios.
- Terminally the relative weights of the livers were lower in all the test groups than in the controls, but there was no evidence of a dose-related response.

HISTOPATHOLOGY: NON-NEOPLASTIC
- The type and incidence of the other non-neoplastic lesions observed were comparable in test and control groups.

HISTOPATHOLOGY: NEOPLASTIC
- The number of lymphoreticular pulmonary tumours in males decreased with increasing levels of sulphite in the diet.
- The incidence of thyroid and pituitary tumours in the control group of the males was exceptionally low, whereas those noted in the various test groups represented numbers normally found in the strain used.
- All other neoplasms occurred in a random manner with no apparent relationsship between number, location of tumours and treatment.

OTHER FINDINGS
- There was a dose-related decrease in the thiamine content of the urine at sulphite levels of 0.125 % and above and in the liver at levels above 0.25 %
- The group given 2 % sulphite showed no distinct changes in the thiamine status when compared with control rats on the stock diet without extra thiamine.

Effect levels

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Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
108 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see "Remarks"
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
> 955 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No signs of systemic toxicity were observed and the NOAEL can be expected above the highest dose of 2% metabisulphite corresponding to 955 mg/kg bw/d Na2S2O5 (or 640 mg/kg bw/d as SO2 equivalents).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Calculation of the dose level in mg/kg bw/d was done by converting % diet into ppm and by assuming an average body weight for older rats of 400 g and a daily feed intake of 20 g as follows:

0.215 % = 2150 ppm x 0.05 = 108 mg/kg bw/d Na2S2O5, equivalent to 72 mg/kg bw/d SO2

1.91% = 19100 ppm x 0.05 = 955 mg/kg bw/dNa2S2O5, equivalent to 640 mg/kg bw/d SO2

Applicant's summary and conclusion

Conclusions:
Based on the occurrence of occult blood in the faeces and changes in gastric morphology at dose levels of 0.5% or more, the NOAEL for local effects in this study is represented by the dose of 0.25% metabisulphite (or 0.215% accounting for the loss of metabisulphite). The corrected dose level corresponded to a dose of 108 mg/kg bw/d Na2S2O5 or an equivalent dose of 72 mg SO2/kg bw/day. Because there were no evidence of systemic toxicity following chronic treatment, the NOAEL for systemic effects can be expected above the highest dose of 2% metabisulphite corresponding to 955 mg/kg bw/d of Na2S2O5 (or 640 mg/kg bw/d as SO2 equivalents).