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Toxicological information

Toxicity to reproduction

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 / 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 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.

Estrous 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 animals)

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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: estrous 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)

open allclose all
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)
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.

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.