Calcium thiosulphate

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Essential details for an assessment are given.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

The Toxicity of Sulphite. I. Long-term Feeding and Multigeneration Studies in Rats.

Author:

Til, H.P., Feron, V.J., deGroot, A.P.

Year:

1972

Bibliographic source:

Fd. Cosmet. Toxicol. 10, 291-310

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

Test material information

Constituent 1

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 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.

Details on mating procedure:

- All rats of the F0-generation were mated within their diet group at about wk 21 and half of them also at wk 34.
- The rats of the F1a-generation were mated at wk 12 and 30 to produce the F2a and F2b litters.
- Ten males and 15 females of the F2a litters were selected for producing the next generation by mating them at wk 14 and 22.
- Group matings were used throughout and lasted for a period of 2 wk.
- 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.

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

Details on study schedule:

- All rats of the F0-generation were mated within their diet group at about wk 21 and half of them also at wk 34.
- Ten male and ten female rats from the first litter of each diet group (F1a-generation) were selected at weaning for rearing further generations.
- All remaining weanling rats were discarded.
- The rats of the F0-generation as well as the selected ras of the F1a-generation were maintained on their diets for a period of 104 weeks.
- The rats of the F1a-generation were mated at wk 12 and 30 to produce the F2a and F2b litters.
- Ten males and 15 females of the F2a litters were selected for producing the next generation by mating them at wk 14 and 22.
- The litters resulting from these matings (F3) were discarded at weaning, whereas their parents were kept on their diets for about 30 wk.
- Group matings were used throughout and lasted for a period of 2 wk.
- At day 20 after the beginning of the mating period, the females were caged individually until after the litters had been weaned.
- Records were made of the number of pups in each litter and of the total weight of the litter at days 1, 8 and 21.
- On the first day, litters containing more than eight pups were randomly reduced in order to equalize the stress of lactation among the dams.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.125% (ca. 50 mg/kg bw)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.25% (ca. 110 mg/kg bw)
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.5 % (ca. 220 mg/kg bw)
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1.0% (ca. 460 mg/kg bw)
Basis:
nominal in diet

Doses / concentrations 5

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 used.

Examinations

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: testis

Litter observations:

Records were made of the number of pups in each litter and of the 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, 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.

Postmortem examinations (offspring):

not specified

Statistics:

no data

Reproductive indices:

see below

Offspring viability indices:

see below

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

occult blood in faeces

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not specified

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)

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.

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.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- The interim results obtained after 1 year did not indicate any effect of sulphite on organ-to-body weigth ratios.
- No effect on the relative weigths of reproductive organs in any of the successive generations were reported.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Pathological changes attributable to the feeding of sulphite were observed only in the stomach.
- No gross pathological changes were reported for reproductive organs.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- 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.
- No histopatholgical changes were reported for reprocuctive organs.

Effect levels (P0)

1

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'

Remarks on result:

other: Generation: F1, F2a and F2b (migrated information)

2

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'

Remarks on result:

other: Generation: F1, F2a, F2b and F3 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

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.
- The number of F2a pups was significantly reduced in groups fed ≥ 0.5% metabisulphite during the first mating cycle. However, there was no tendency towards a decrease with increasing dietary sulphite levels and moreover this reduction did not occur in the second litters (F2b).
- During the lactation period the body weights of the young in the 2% group were generally lower than those of the controls.
- Dietary levels of 1% or below were sometimes associated with significantly decreased body weights on days 8 or 21, but there was no distinct dose-related response.

Effect levels (F1)

Remarks on result:

not determinable

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

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.