Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
three-generation reproductive 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:
Essential details for an assessment are given.
Justification for type of information:
Read-across
The basis for the read-across concept for this project is the equilibrium between sulfites, hydrogensulfites, and metabisulfites in aqueous solutions depending on pH value which is clearly described in published literature and summarised in the following equations:[1],[2]
SO2 + H2O <->`H2SO3´ H2SO3<->H+ + HSO3- <-> 2H+ +SO32- 2HSO3- <->H2O +S2O52 –
As the nature of the cation should make no significant difference in this case concerning toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anion are considered relevant. Based on the described equilibrium correlations, we propose unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites. Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II) so that this substance can also be added to the read-across concept.[2],[1]
It is expected for this case that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines and so the products of decomposition have to be considered.
2 S2O42-+ H2O→2HSO3-+ S2O32 -

All sulfite, hydrogensulfite and metabisulfite substances are highly soluble in water, establishing upon dissolution an equilibrium that depends on solution pH as follows: ,

1. SO2 + H2O <-> H2SO3
2. H2SO3 <-> H+ + HSO3- <-> 2H+ + SO32-
3. 2 HSO3- <-> H2O + S2O52-

Under oxidising conditions, e.g., in surface waters, sulfite is oxidized to sulfate catalytically by (air) oxygen or by microbial action. A half-life of 77 hour was measured in deionized water, already suggesting substantial abiotic degradation. However, the presence of metal cations in the environment, such as copper, iron and manganese, accelerates the oxidation rate. In soils, HSO3- and SO32- ions are unstable and quickly oxidise. Further, because of the instability of SO32-, metal sulfites are generally too soluble to persist in soils. Thus, the most stable and predominant sulfur form in freshwater and in all but highly reduced environments is sulfate (SO42-). In highly reduced soils and sediments, sulfites may be reduced to sulfides (Lindsay, 1979; OECD SIDS, 2012).

Only the properties of the sulfite anion are considered relevant determinants of environmental toxicity since respective cations, i.e. ammonium, calcium, magnesium, sodium and potassium, are not assumed to contribute substantially to differences therein. Sulfite, although naturally present in the environment and also a metabolite and intermediate of sulfur-containing amino acids in organisms, may have an impact on the environment at elevated levels. Sulfites do not bioaccumulate.

In sum, unrestricted read-across between the sulfites, hydrogensulfites and metabisulfites is justified.
Cross-referenceopen allclose all
Reason / purpose:
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.
Reason / purpose:
reference to same study
Reason / purpose:
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 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:
reference to same study
Reference
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:
Read-across
The basis for the read-across concept for this project is the equilibrium between sulfites, hydrogensulfites, and metabisulfites in aqueous solutions depending on pH value which is clearly described in published literature and summarised in the following equations:[1],[2]
SO2 + H2O <->`H2SO3´ H2SO3<->H+ + HSO3- <-> 2H+ +SO32- 2HSO3- <->H2O +S2O52 –
As the nature of the cation should make no significant difference in this case concerning toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anion are considered relevant. Based on the described equilibrium correlations, we propose unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites. Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II) so that this substance can also be added to the read-across concept.[2],[1]
It is expected for this case that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines and so the products of decomposition have to be considered.
2 S2O42-+ H2O→2HSO3-+ S2O32 -

All sulfite, hydrogensulfite and metabisulfite substances are highly soluble in water, establishing upon dissolution an equilibrium that depends on solution pH as follows: ,

1. SO2 + H2O <-> H2SO3
2. H2SO3 <-> H+ + HSO3- <-> 2H+ + SO32-
3. 2 HSO3- <-> H2O + S2O52-

Under oxidising conditions, e.g., in surface waters, sulfite is oxidized to sulfate catalytically by (air) oxygen or by microbial action. A half-life of 77 hour was measured in deionized water, already suggesting substantial abiotic degradation. However, the presence of metal cations in the environment, such as copper, iron and manganese, accelerates the oxidation rate. In soils, HSO3- and SO32- ions are unstable and quickly oxidise. Further, because of the instability of SO32-, metal sulfites are generally too soluble to persist in soils. Thus, the most stable and predominant sulfur form in freshwater and in all but highly reduced environments is sulfate (SO42-). In highly reduced soils and sediments, sulfites may be reduced to sulfides (Lindsay, 1979; OECD SIDS, 2012).

Only the properties of the sulfite anion are considered relevant determinants of environmental toxicity since respective cations, i.e. ammonium, calcium, magnesium, sodium and potassium, are not assumed to contribute substantially to differences therein. Sulfite, although naturally present in the environment and also a metabolite and intermediate of sulfur-containing amino acids in organisms, may have an impact on the environment at elevated levels. Sulfites do not bioaccumulate.

In sum, unrestricted read-across between the sulfites, hydrogensulfites and metabisulfites is justified.
Reason / purpose:
reference to same study
Reason / purpose:
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:
not specified
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals 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
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
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
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
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.
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).
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 = 955 mg/kg bw/dNa2S2O5, equivalent to 640 mg/kg bw/d SO2

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).

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

Reference
Name:
Unnamed
Type:
Constituent
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 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
Vehicle:
unchanged (no vehicle)
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%.
- 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.

Details on mating procedure:
- All rats of the F0-generation were mated within their diet group at about week 21 (producing F1a) and half of them also at week 34 (producing F1b).
- The rats of the F1a-generation were mated at week 12 and 30 to produce the F2a and F2b litters, respectively.
- Males and females of the F2a litters were selected for producing the next generation by mating them at week 14 and 22.

- Group matings were used throughout and lasted for a period of 2 weeks.
- At day 20 after the beginning of the mating period, the females were caged individually until after the litters had been weaned.
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:
F0-Generation: 104 weeks (21 weeks or 34 weeks before mating and up to a total of 104 weeks)
F1a-Generation: 104 weeks (12 weeks or 30 weeks before mating and up to a total of 104 weeks)
F2a-Generation: ca. 30 weeks (14 weeks or 22 weeks before mating and up to a total of about 30 weeks)
Frequency of treatment:
continuously
Details on study schedule:
not specified
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:
F0 - Generation: 20 males / 20 females
F1a - Generation: 10 males / 10 females
F2a - Generation: 10 males / 15 females
Control animals:
yes, plain diet
Details on study design:
not specified
Positive control:
not specified

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs and mortality
At week 32, 64 and 100 all rats of the F0- and F1a-Generations and at week 28 those of the F2a-Generation 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.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
F0-Generation: week 52, 78, and 100
F1a-Generation: week 52, and 102
F2a-Generation: week 20
- 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 (F0-Generation only)
- 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:
F0-Generation: week13, 28, 52, 78, and 101
F1a-Generation: week 28, 52, and 100
F2a-Generation: week 28
- 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.

FURTHER 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.
Oestrous cyclicity (parental animals):
not specified
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations: testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed: yes, on the first day, the litters containing more than eight pups were randomly reduced to that number in order to equalize the stress of lactation among the dams.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F3 offspring:
- number of pups in each litter
- total weight of the litter at days 1, 8 and 21.
Postmortem examinations (parental animals):
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 (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.
Postmortem examinations (offspring):
not specified
Statistics:
not specified
Reproductive indices:
not specified
Offspring viability indices:
not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not specified
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
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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- The results of the matings in successive generations showed no consistent differences between groups for fertility and the number of pups/litter.

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 (P0)

open allclose all
Key result
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'
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
> 955 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

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 13-40% of the animals on diets containing 1 % sulphite.
Dermal irritation (if dermal study):
not specified
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:
not specified
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):
- lesions occurred in both the fore- and glandular stomach and were mainly characterized by either hyperplasia or inflanunation. 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, except in the case of males of the F1-Genertion given 2 % sulphite. However, no deaths occurred in the females of the same group.

BODY WEIGHT AND WEIGHT GAIN
- There was a marginal reduction in body-weight gain in both sexes of the F1- Generation rats given 2 % sulphite. This occurred also in females given 0.125, 0.25 and 0.5% sulphite in the F1-generation, but there was no dose-relationship and, moreover, the differences could partly be explained by the higher initial body weights of the controls.

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

HAEMATOLOGY
- Males at 2 % showed an increase in leucocyte count at wk 102.
- Otherwise, all haematological values for test and control animals were within normal ranges at all stages.

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
- The interim results obtained after 1 yr 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.
- sulphite feeding had no apparent effect on the relative weights of any other organs.

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 the glandular stomach were seen grossly in the groups given the two highest sulphite levels.

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 Iymphoreticular 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 male rats was exceptionally low, whereas those noted in the various test groups represented numbers normally found in the strain of rats used.
- All other neoplasms occurred in a random manner with no apparent relationship between number, location or type of tumours and the treatment.

OTHER FINDINGS
- There was a dose-related decrease in the thiamine content of the urine at sulphite levels of 0.125 % and above
- However, 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.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P1)

REPRODUCTIVE PERFORMANCE
- The results of the matings showed no consistent differences between groups in female fertility

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
local toxicity (P1-Generation)
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:
fertility (P1-Generation)
Effect level:
> 955 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:
local toxicity (P2-Generation)
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:
fertility (P2-Generation)
Effect level:
> 955 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see "Remarks"

Target system / organ toxicity (P1)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- during the lactation period the body weights of the young in the 2 % group were generally lower than those of the controls and the lower sulphite groups.
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 specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

- No consistent differences between groups were observed for the number of pups/litter, birth weight or mortality of the offspring.



Effect levels (F1)

Remarks on result:
other: there was a slight growth retardation during lactation in offspring of the 2% group.

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- during the lactation period the body weights of the young in the 2 % group were generally lower than those of the controls and the lower sulphite groups.
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 specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not specified

Details on results (F2)

- No consistent differences between groups were observed for the number of pups/litter, birth weight or mortality of the offspring.
- dietary levels of 1 % or below were sometimes associated with significantly decreased body weights at days 8 and 21, but there was no distinct dose-related response.

Effect levels (F2)

Remarks on result:
other: here was a slight growth retardation during lactation in offspring of the 2% group.

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive 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

P2 (third parental generation)

 

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.

- All rats in the highest dose group showed indications of occult blood in the faeces, while this effect occurred in only 20-30% of the animals on diets containing 1 % sulphite.

 

BODY WEIGHT AND WEIGHT CHANGES

- There was a marginal reduction in body-weight gain in both sexes of the F2-generation rats given 2 % sulphite.. This occurred also in females given 0·25 and 0·5 % in the F2-generation, but there was no dose-relationship and, moreover, the differences could partly be explained by the higher initial body weights of the controls.

 

FOOD CONSUMPTION AND COMPOUND INTAKE

- There were no distinct differences in food consumption, but a slight reduction was observed in the 2 % group of the F2-Generation during the first 2 wk of the experiment.

 

HAEMATOLOGY

- all haematological values for test and control animals were within normal ranges at all stages.

 

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

- The interim results obtained after 1 yr did not indicate any effect of sulphite on organ-to-body weight ratios.

- No distinct decrease in liver weight was observed.

- The relative weight of the kidneys was slightly, but significantly (P < 0·05), increased in the 2 % females.

- Sulphite feeding had no apparent effect on the relative weights of any other organs in any of the successive generations.

 

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 the glandular stomach were seen grossly in the groups given the two highest sulphite levels.

 

HISTOPATHOLOGY: NON-NEOPLASTIC

- lesions occurred in both the fore- and glandular stomach and were mainly characterized by either hyperplasia or inflanunation. They were either present only at the 1 and 2 % dosage levels or their incidence was distinctly increased at these levels.

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

 

REPRODUCTIVE PERFORMANCE

- The results of the matings showed no consistent differences between groups in female fertility

 

OTHER FINDINGS

- There was a dose-related decrease in the thiamine content of the urine at sulphite levels of 0.125 % and above.

- However, 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.

 

 

F3 generation

- No consistent differences between groups were observed for the number of pups/litter, birth weight or mortality of the offspring.

- A significant reduction in the number of young was observed with 0·5, 1 and 2 % sulphite. However, there was no tendency towards a decrease with increasing dietary levels and moreover this reduction didnot occur in the second litters.

- during the lactation period the body weights of the young in the 2 % group were generally lower than those of the controls and the lower sulphite groups.

- dietary levels of 1 % or below were sometimes associated with significantly decreased body weights at days 8 and 21, but there was no distinct dose-related response.

 

Applicant's summary and conclusion

Conclusions:
The most sensitive parameters of chronic treatment of rats wit Na2S2O5 was the occurrence of occult blood in the faeces and changes in gastric morphology at dose levels of 0.5% or more. Thus, the NOAEL for local effects is represented by the dose of 0.25% metabisulphite (or 0.215% accounting for the los/s of metabisulphite). The corrected dose level corresponds to a dose of 108 mg/kg bw/d Na2S2O5 or an equivalent dose of 72 mg SO2/kg bw/day.
Based on the results of this study, no evidence of a treatment-related effect on reproduction and fertility was seen. Thus, the NOAEL for fertility can be expected above a dose level of 2% metabisulfite, corresponding to a dose of 955 mg/kg bw/d Na2S2O5 or 640 mg SO2/kg bw/day. However, there was a slight growth retardation during lactation in offspring of the 2% group.