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Genetic toxicity: in vivo

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in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
The positive findings appear plausible. However there are reasons to question these findings: (i) silver staining not specific for DNA, since silver cations also interact with all negatively charged functional groups in biomolecules, such as proteins, DNA, RNA. It is therefore unclear whether the measured tail was specific to DNA damage. A DNA-specific fluorophore was not used. (ii) DNA damage is usually expressed either in tail length, DNA content in tail or tail moment. Crude visual inspection is prone to subjective interpretation by the experimenter which may result in a significant intra-day and day to day variation. The metric for DNA damage should be measured using (i) micrometer in eyepieces (ii) ruler on photographs or (iii) a computerised automated system, so ensure consistent and reproducible interpretation of DNA tails. Due to the mentioned ambiguities, it is questionable whether a true substance induced DNA damage was observed in the comet assay.
Justification for type of information:
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.

Data source

Reference Type:
Genotoxicity of sodium metabisulfite in mouse tissues evaluated by the comet assay and the micronucleus test
Carvalho, I.M.C.M.M. et al.
Bibliographic source:
Mutation Research 720: 58 - 61.

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
In this study the genotoxic effect of sodum metabisulfite (concentrations: 0.5, 1, or 2 g/kg bw) on different tissues of the mouse was investigated by use of the comet assay (liver and blood cells).
GLP compliance:
not specified
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium disulphite
EC Number:
EC Name:
Disodium disulphite
Cas Number:
Molecular formula:
disodium disulphite
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): sodium metabisulfite (purchased Merck)
- Molecular formula: Na2S2O5

Test animals

Details on test animals or test system and environmental conditions:
TEST ANIMALS - outbred CF1 mice
- Source: Federal University of Piauí (UFPI), Teresina, PI, Brazil
- Age at study initiation: 5–7 weeks old
- Weight at study initiation: 20–40 g
- Diet (ad libitum): commercial standard mouse cube diet (Labina-PURINE)
- Water (ad libitum)

- Temperature: approx. 24°C
- Relative humidity: approx. 60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: water
Details on exposure:
The test substance was dissolved in the vehicle (solution pH∼5) immediately before administration by gavage.
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 other: g/kg bw
actual ingested
Dose / conc.:
1 other: g/kg bw
actual ingested
Dose / conc.:
2 other: g/kg bw
actual ingested
No. of animals per sex per dose:
5 females/5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
- Route of administration: gavage
- Doses / concentrations: 25 mg/kg


Tissues and cell types examined:
To calculate a damage index (DI), cells were visually allocated into five classes according to tail size (0 = no tails and 4 =maximum-length tails), which resulted in a single DNA damage score for each sample and consequently for each group studied. Thus, the damage index (DI) of the group could range from 0 (completely undamaged = 100 cells × 0) to 400 (maximum damage = 100 cells × 4). The damage frequency (DF in %) was calculated for each sample, based on the number of cells with tail versus those without. All sides were coded for blind analysis.
Details of tissue and slide preparation:
To determine the maximum tolerated dose, six animals (three of each gender) were used per dose. Groups received 0.5, 1, or 2 g sodium metabisulfite per kg (0.1 mL/10 g body weight). Animals were observed during a 24-hour period and then sacrificed by cervical dislocation. Mortality and clinical signs of toxicity were not observed in this preliminary study.

The analysis was conducted according to the protocol of Singh et al. (1988)*, with some modifications [Da Silva et al., 2000; Tice, 1995]*. Cells from different tissues were obtained according to the method of Tice (1995)*. Bone marrow perfusion was performed on the femur with 2 mL cold RPMI medium, and each piece of liver was placed in cold RPMI (0.5 mL) and finely minced, in order to obtain a cell suspension. Blood cells (whole blood with heparin) and cells isolated from tissues (10 µL aliquots) were embedded in low melting point agarose (0.75%, w/v, 90µL) and the mixture was added to a microscope slide pre-coated with normal melting point agarose (1.5%, w/v) and covered with a coverslip. The slide was briefly placed on ice for the agarose to solidify and the coverslip was carefully removed. Next, the slide was immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10.0–10.5). The slides were incubated in freshly made alkaline buffer (300 mM NaOH, 1 mM EDTA, pH> 13) for 20 minutes for DNA unwinding, and electrophoresis was performed in the same buffer. Electrophoresis was performed for 15 minutes at 300 mA and 25 V (0.7 V/cm). All these steps were carried out under dim indirect light. Following electrophoresis, slides were neutralized in 400 mM Tris (pH 7.5) and fixed (15%, w/v, trichloroacetic acid, 5%, w/v, zinc sulfate, 5% glycerol), washed in distilled water, and dried overnight.

The gels were re-hydrated for 5 minutes in distilled water, and then stained for 15 minutes (37°C) with a solution containing the following sequence: 34mL solution B (0.2%, w/v,ammonium nitrate, 0.2%, w/v, silver nitrate, 0.5%, w/v, tungstosilicic acid, 0.15%, v/v, formaldehyde, 5%, w/v, sodium carbonate) and 66 mL solution A (5% sodium carbonate). The staining was stopped with 1% acetic acid and the gels were air-dried [Nadin et al., 2001]*.

- N.P. Singh, M.T. McCoy, R.R. Tice, E.L.A. Scheider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191.
- J. Da Silva, T.R.O. Freitas, J.R. Marinho, G. Speit, B. Erdtmann, Effects of chronic exposure to coal in wild rodents (Ctenomys torquatus) evaluated by multiple methods and tissues, Mutat. Res. 470 (2000) 39–51.
- R.R. Tice, Applications of the single cell gel assay to environmental biomonitoring for genotoxic pollutants, in: B.E. Butterworth, L.D. Corkum, J. Guzmán-Rincón (Eds.), Biomonitors and Biomarkers as Indicators of Environmental Change, Plenum Press, New York, 1995, pp. 69–79.
- S.B. Nadin, L.M. Vargas-Roig, D.R. Ciocca, A silver staining method for single-cell gel assay, J. Histochem. Cytochem. 49 (2001) 1183–1186.
Evaluation criteria:
not specified
The normality of variables was evaluated using the Kolmogorov–Smirnov test. The statistical differences between the groups were analyzed using the nonparametric two-tailed Kruskal–Wallis Test with the Dunn correction for multiple comparisons for comet assay results. Difference between genders was tested using the Wilcoxon–Mann–Whitney test. The critical level for rejection of the null hypothesis was P = 5%.

Results and discussion

Test results
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- positive control showed significant values in mean damage index and damage frequency as compared to negative control.
- sodium metabisulfite group: a significant increase was observed on both damage index and damage frequency values, when comparing 1 and 2 g/kg doses to negative control, for all tissues.

Applicant's summary and conclusion

According to the authors, 1 and 2 g/kg sodium metabisulfite significantly induced DNA damage, as shown by the comet assay in mouse blood, liver, and bone marrow cells.