Sodium sulphite

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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:

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

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 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

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

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

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

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: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

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.

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'

Critical effects observed:

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 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: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

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

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"

Critical effects observed:

not specified

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

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

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

Remarks on result:

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

Critical effects observed:

not specified

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

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

- 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.

Remarks on result:

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

Critical effects observed:

not specified

Reproductive 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

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.

 

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

Read-across concept for sulfites, hydrogensulfites, metabisulfites, dithionites and thiosulfates:

A comprehensive read-across concept has been developed for sulfites, hydrogensulfites and metabisulfites, based on the pH-dependant equilibrium in aqueous solutions which is summarised in the following equations:^[1],[2]

           SO₂+ H₂O <->`H₂SO₃´         H₂SO₃<->H⁺+ HSO₃^-<->2H⁺+SO₃^2-    2HSO₃^-<->H₂O +S₂O₅^2-

Since the nature of the cation (i.e., sodium, potassium, ammonium…) is not assumed to contribute substantially to differences in toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anion are considered as relevant determinants. Based on the described equilibrium correlations, unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites is considered justified.

 

Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II)^2,[1], so that this substance can also be considered to be covered by the read-across concept described above. Since it can easily be anticipated that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines, instead the products of decomposition have to be considered:

 

       2 S₂O₄^2-+ H₂O→2HSO₃^-+ S₂O₃^2-

 

Not fully covered by this read-across concept is the substance class of thiosulfates: although the thiosulfates are also well known to disproportionate in aqueous solution to form polythionic acids and SO₂(HSO₃^-), this requires somewhat different, more acidic conditions. Therefore, read-across to sulfites is primarily restricted to appropriate physiological conditions, i.e. oral administration where the gastric passage with the strongly acidic conditions in the stomach will facilitate the chemical disproportionation described above:

 

       HS₂O₃^-+ H₂S₂O₃→HS₃O₃- + SO₂+ H₂O

[1]Hollemann Wiberg, Lehrbuch der Anorganischen Chemie, 101.Auflage

[2]Handbook of Chemistry and Physics, Ed. Lide, DR, 88^thedition, CRC Press

One key study (Til 1972) and one supportive study (Lockett 1960) were identified to evaluate the effects sodium metabisulphite on fertility and reproduction. In both studies, male and female rats received different dietary dose levels of up to 2% Na₂S₂O₅for periods of about 2 years over three successive generations. The results of these studies did not show evidence of a treatment-related effect on fertility and reproduction. Thus, the NOAEL for fertility can be expected above the highest dose of 2% sodium metabisulphite, corresponding to 955 mg/kg bw/d of Na₂S₂O₅.

However, there was a slight growth retardation during lactation in offspring of the highest dose level of 2%, corresponding to a dose of 955 mg/kg bw/d Na₂S₂O₅.

Short description of key information:
One key study (Til 1972) and one supportive study (Lockett 1960) were identified to evaluate the effects sodium metabisulphite on fertility and reproduction. As a result, the NOAEL for fertility can be expected above the highest dose of 2% sodium metabisulphite, corresponding to 955 mg/kg bw/d of Na2S2O5.

Effects on developmental toxicity

Description of key information

The following developmental toxicity studies were identified as key studies:
- sodium metabisulphite in rabbits (NTIS_1974)
- potassium metabisulphite in rats (Anonymous_1975)
- potassium metabisulphite in rats (Ema_1985) - diet study
- potassium metabisulphite in mice ((Anonymous_1975)
- sodium sulphite heptahydrate in rats (Itami_1989) - diet study
- sodium bisulphite in mice, rats and hamsters (NTIS_1972)
- sodium bisulphite in rabbits (NTIS_ 1974)
- sodium thiosulphate in mice, rats and hamsters (Anonymous_1972)
- sodium thiosulphate in rabbits (Anonymous_1974)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

Read-across concept for sulfites, hydrogensulfites, metabisulfites, dithionites and thiosulfates:

A comprehensive read-across concept has been developed for sulfites, hydrogensulfites and metabisulfites, based on the pH-dependant equilibrium in aqueous solutions which is summarised in the following equations:^[1],[2]

           SO₂+ H₂O <->`H₂SO₃´         H₂SO₃<->H⁺+ HSO₃^-<->2H⁺+SO₃^2-    2HSO₃^-<->H₂O +S₂O₅^2-

Since the nature of the cation (i.e., sodium, potassium, ammonium…) is not assumed to contribute substantially to differences in toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anion are considered as relevant determinants. Based on the described equilibrium correlations, unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites is considered justified.

 

Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II)^2,[1], so that this substance can also be considered to be covered by the read-across concept described above. Since it can easily be anticipated that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines, instead the products of decomposition have to be considered:

 

       2 S₂O₄^2-+ H₂O→2HSO₃^-+ S₂O₃^2-

 

Not fully covered by this read-across concept is the substance class of thiosulfates: although the thiosulfates are also well known to disproportionate in aqueous solution to form polythionic acids and SO₂(HSO₃^-), this requires somewhat different, more acidic conditions. Therefore, read-across to sulfites is primarily restricted to appropriate physiological conditions, i.e. oral administration where the gastric passage with the strongly acidic conditions in the stomach will facilitate the chemical disproportionation described above:

 

       HS₂O₃^-+ H₂S₂O₃→HS₃O₃- + SO₂+ H₂O

[1]Hollemann Wiberg, Lehrbuch der Anorganischen Chemie, 101.Auflage

[2]Handbook of Chemistry and Physics, Ed. Lide, DR, 88^thedition, CRC Press

In none of the studies conducted in preganat rats, mice, hamsters and rabbits with oral administration of sodium bisulfite, sodium metabisulfite, potassium metabisulfite or sodium thiosulfate by gavage during the phase of organogenesis clear evidence of discernible effects on nidation or on maternal or foetal survival were observed. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls. Thus, the NOAELs for maternal and developmental toxicity can be expected above the highest dose levels investigated:

- sodium metabisulfite in rabbits: > 123 mg/kg bw/d

- potassium metabisulfite in rats: > 155 mg/kg bw/d

- potassium metabisulfite in mice: > 125 mg/kg bw/d

- sodium bisulfite in mice: > 150 mg/kg bw/d

- sodium bisulfite in rats: > 110 mg/kg bw/d

- sodium bisulfite in hamsters: > 120 mg/kg bw/d

- sodium bisulfite in rabbits: > 100 mg/kg bw/d

- sodium thiosulfate in mice: > 550 mg/kg bw/d

- sodium thiosulfate in rats: > 400 mg/kg bw/d

- sodium thiosulfate in hamsters: > 400 mg/kg bw/d

- sodium thiosulfate in rabbits: > 580 mg/kg bw d

In two other studies with potassium metabisulfite and sodium sulfite heptahydrate in rats given at high dietary dose levels during the phase of organogenisis, maternal toxicity evidenced by reduction in maternal body weight gain during pregnancy and food intake was observed in the highest dose groups, resulting in NOAELs of 1320 mg/kg bw/d for potassium metabisulfite or 850 mg/kg bw/d for sodium sulfite heptahydrate.

The postnatal survival rate of the offspring of the 10% potassium metabisulfite group was slightly decreased, most probably related to maternal malnutrition. Thus, the NOAEL for foetotoxicity was established at a dose level of 1320 mg K₂S₂O₅/kg bw/d. There was no clear evidence for foetal toxicity in the study conducted with sodium sulfite heptahydrate, but slight foetal growth retardation was observed in all dose groups (at and above approx. 100 mg/kg bw/d Na₂SO₃x 7 H₂O). However, there was no clear dose-relationship and this effect was not observed in the live-birth part of the study and was therefore not considered as of toxicological relevance.

External, skeletal and internal examinations of the foetuses revealed no evidence of teratogenesis in any of the groups.

Justification for classification or non-classification

All available animal data did not show any evidence of effects on fertility, toxicity to reproduction, developmental toxicity or teratogenicity of sodium metabisulfite or any similar substance within the read-across concept for the group of sulfite substances. Thus, based on the lack of any effects on reproductive performance and organs, the reproductive tract is not considered to represent a target organ of toxicity.

Therefore the requirements according to regulation (EC) 1272/2008 for classification for the hazard class Reproductive Toxicity are not fulfilled andclassification is not required for sodium sulfite and any other of the sulfite substances.