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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
other: combined repeated dose and reproduction toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
no data
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.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

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:
no
Limit test:
no

Test material

Constituent 1
Reference substance name:
Disodium disulphite
EC Number:
231-673-0
EC Name:
Disodium disulphite
Cas Number:
7681-57-4
IUPAC Name:
disodium disulphite
Constituent 2
Reference substance name:
disodium disulfite
IUPAC Name:
disodium disulfite
Details on test material:
- Name of test material (as cited in study report): sodium metabisulfite
- Molecular formula (if other than submission substance): Na2S2O5
- Molecular weight (if other than submission substance): 190.1 g/mol
- Substance type: technical product
- Physical state: solid, white powder
- Analytical purity: between 95 and 99%
OTHER
- Samples obtained from Amsterdamsche Chinine Fabriek (ACF), Amsterdam, Netherlands
No further details are given.

Test animals

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.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
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%. 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)
Doses / concentrationsopen allclose all
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 positive control substance was tested.

Examinations

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

Results and discussion

Results of examinations

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 examined
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. metabisulphite.

Effect levels

open allclose all
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'
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).