Disodium 3,3'-dithiobis[propanesulphonate]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Acceptable well documented publication which meets basic scientific principles, read-across

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
For details, please refer to the attached read-across justification. In brief:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
There are two category approaches relevant for all human health associated endpoints: Chain-length category and similar metabolic pathway.
Chain-length Category: Both SPS and Dimesna are sodium salts of two sulphonated alkanes connected via a disulphide group. SPS contains two propane moieties, Dimesna ethane ones, hence, these chemicals only differ minor in their hydrocarbon chains in one –CH2– group. The same applies to the carbon chain in MPS and MESNA, connecting the sodium sulfonate with the sulfhydryl moiety. The reactivity and toxicological relevance of this difference in chain length is considered to be minor compared to the chemicals properties triggered by the two remaining respective functional groups. Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the hydrocarbon chain length becomes obvious. The melting points for the disulphide compounds and the sulfhydryl ones are consistent, and the ones for the ethane derivatives are as expected slightly lower. All compounds are very soluble in water, and similar consistencies are noted with regard to vapour pressure and partition coefficient.
Metabolic pathway: Here it is aimed to justify the read-across from both MPS to SPS and Mensa to Dimesna, and Dimesna to SPS and Mesna to MPS (and vice versa) based on the available information on their metabolism.
Generally, Mesna and Dimesna are considered to be a metabolite of each other. Also, other metabolites of Mesna were identified, besides Mesna-Mesna (i.e., Dimesna), such as Mesna-Cys, Mesna-homocysteine, Mesna-cysteinylglutamate, Mesna-cysteinylglycine, and Mesna-GSH which have been collectively termed “Dimesna” in some studies, while others refer to the mixed disulfides containing a single Mesna moiety as “Mesna”, quantifying Dimesna separately. The relevant functional groups for the enzymatic and non-enzymatic metabolism of Dimesna and Mesna are the disulphide resp. thiol functional groups. Those are both contained in the related substances SPS and MPS, which only differ from the former in their hydrocarbon chains in one –CH2– group, the basic structure and functional groups are however identical. Hence, only taking into account the given functional groups, a similar toxicodynamic behaviour of SPS and MPS compared to Dimesna and Mesna can be expected.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
As shown above, Mesna, MPS, Dimesna and SPS can be used for read-across to each other by grouping of chemicals. Mesna and MPS and Dimesna and SPS share similar physico-chemical properties as well as they exhibit similar toxicological properties, where data is available. Their alkyl side chains differ only in one –CH2- group, so it can be concluded that e.g. absorption, distribution patterns, or excretion from organ systems and body are comparable. Furthermore, Dimesna and Mesna are considered a metabolite of each other, which allows the conclusion that the same also applies for SPS and MPS. Conclusively, data for Mesna, MPS, and Dimesna can be used to cover data gaps for SPS; especially for the required endpoints for human health assessment.
Freely available toxicological information on Dimesna is lacking, so the available information on SPS, MPS and Mesna will be compared in order to obtain contributing information for the read-across justification, as set out in the attachment
The available data indicate that SPS, MPS and Mesna do not need to be classified as acute toxic according to Regulation (EC) No 1272/2008, all available LD50 (oral or dermal) values are greater than 2000 mg/kg bw, clearly indicating the comparability of the substances and the relative harmlessness of all group members including the target chemical SPS with regard to acute toxicity.
All available Ames tests on SPS, MPS and Mesna are consistently negative. Furthermore, the available in vitro micronucleus test on MPS, the SCE assay and in vivo micronucleus test on Mesna do also not give any indication that this group of substances bears any genotoxic properties. Here, gene mutation as well as chromosome mutation and clastogenicity endpoints are covered. In addition, the SCE assay is indicative for an enhanced repair activity upon genotoxic damage, which may result in several outcomes, e.g. point mutations, chromosome breaks etc., which support additionally the hypothesis that this group does not bear genotoxic properties of any kind.
In the available publications on carcinogenicity, both Mesna and its dimer Dimesna did not trigger any signs of toxicity or carcinogenic activity up to the highest dose tested, i.e., 15 resp. 35 mg/kg bw/d with lifetime exposure. Data on Mesna alone indicate that both doses could have been chosen much higher without resulting in any effects, as e.g. a NOAEL was determined to be 350 mg/kg bw/d over an exposure period of 39 weeks. Again this indicates that this group of chemicals does not trigger any relevant adverse effects upon repeated dosage. Last but not least, Mesna was not identified to be a developmental and or reproductive toxicant in several available studies on that endpoint, up to and including limit dosages of 2000 mg/kg bw/d.

Data source

Reference

Reference Type:

publication

Title:

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo

Author:

Laehdetie, J., Raety, R., and Sorsa, M.

Year:

1990

Bibliographic source:

Mutation Research, 245 (1990) 27-32

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

Principles of method if other than guideline:

Adult male Wistar rats received cyclophosphamide (30 mg/kg bw) and/or Mesna (15 mg/kg bw) intraperitoneally. Both chemicals were dissolved in physiological saline. With regard to Mesna, physiological saline was the negative control and cyclophosphamide was the positive control. The animals were killed 30 hours after the injections and bone marrows were removed and analysed for presence of micronuclei. Micronuclei were scored in May-Grünwald-Giemsa-stained and fluorescent preparations. The proportion of polychromatic erythrocytes (PCE) was determined per 1000 normochromatic erythrocytes (NCE), and the frequency of Micronuclei was counted in 1000 NCEs per animal.

GLP compliance:

no

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3,3'-dithiobis[propanesulphonate]

EC Number:

248-324-3

EC Name:

Disodium 3,3'-dithiobis[propanesulphonate]

Cas Number:

27206-35-5

Molecular formula:

C6H14O6S4.2Na

IUPAC Name:

disodium 3,3'-disulfanediyldipropane-1-sulfonate

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Laboratory Animal Center, University of Kuopio (Finland).
- Weight at study initiation: 250 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: in metabolic cages (2 rats per cage)
- Diet (e.g. ad libitum): the rats had access only to 2% saccharose water, 100 mL per cage during the experiment.
- Water (e.g. ad libitum): no, 100 mL of 2% saccharose water per cage

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: the chemicals are well soluble in physiolical saline
- Concentration of test material in vehicle: 3 g/L (the injected volume was 5 mL/kg resulting in dose of 15 mg/kg bw )

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the injected volume was 5 mL/kg resulting in 15 mg/kg bw of Mesna

Duration of treatment / exposure:

The animals were killed 30 hours after the injections.

Frequency of treatment:

single injections

Post exposure period:

30 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
15 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

not reported

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): cyclophosphamide and its metabolites are the target substances tested in this study. Mesna was tested for its ability to reduce cytotoxicity and mutagenic activity induced by the antitumor drug and the metabolites. Thus, Mesna was injected together with cyclophosphamide to rats. On the other hand, it served as Mesna control, which is here relevant for the assessment of genotoxicity.
- Route of administration: intraperitoneal
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow, PCEs and NCEs.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
One femur of each rat was cleaned for bone marrow micronucleus analyses (Schmid, 1976). 1.5 mL fetal calf serum was injected into the bone marrow to release the contents into test tubes already containing 3.5 mL fetal calf serum. The cells were smeared on clean slides after suspension by Pasteur pipetting, air-dried, fixed in methanol for 10 min and air-dried.

METHOD OF ANALYSIS:
Since it is specially recommended for rat bone marrow MN analyses where Giemsa staining may yield high frequencies of basophilic granules, the double fluorescent staining by Hoechst 33258 and Pyronin Y (MacGregor et al., 1983) was used. MN were scored in both May-Grünwald-Giemsa-stained and fluorescent stained preparations.

One smear per animal was stained with 50% May-Grünwald solution for 3 min, rinsed in distilled water for 1 min, stained with 10% Giemsa for 10 min and rinsed several times with distilled water until the optimal differentiation between polychromatic (PCE) and normochromatic erythrocytes (NCE) was achieved. Another smear was stained in a foil-wrapped jar containing 60 mL phosphate-buffered saline (PBS), 400 µL Hoechst 33258 (1.43 µg/mL), and 600 µL Pyronin Y (Merck, Darmstadt, F.R.G.) for 1 h at room temperature. Pyronin Y (1000 µg/mL aqueous stock solution) was extracted with chloroform 10 times, and the absorbance of the 1:10 working solution was determined at 545 nm. The slides were rinsed with sterile PBS and sterile water and mounted with sterile water. All solutions were filtered through 0.22 µm filters. Only a few slides were stained at a time.

Scoring was performed on coded slides. In May-Grünwald-Giemsa preparations the proportion of PCEs was determined per 1000 NCEs, and the frequency of MN was counted in 1000 PCEs and 1000 NCEs per animal. In Hoechst-Pyronin Y-stained preparations the total number of unnucleated cells (erythrocytes) in each visual field was determined first by phase contrast. Secondly the PCEs were visualized and counted by N2 filter block in a Leitz epi-illumination fluorescence microscope. Thirdly, with filter block A the nuclear fluorescence was visualized. Always when a MN was seen, it was checked whether it was inside a PCE. A total of 1000 PCEs was scored per animal. 12.5 × magnifying oculars and 63 × dry objectives were used.

Evaluation criteria:

Positive response: dose-related increase of MN in PCEs and significant decrease in PCEs/NCEs ratio compared to control.

Statistics:

Statistical analysis of mutagenicity assays was done using Student's t-test. MN results were analysed based on the Poisson distribution (Laehdetie and Parvinen, 1981). Results of PCEs/NCEs ratio were analysed by 2-way analysis of variance with repeated measurements by taking the treatment as a grouping factor and the staining method as the within factor. Pairwise comparisons were performed by Student's t-test with Bonferroni's correction. A BMDP program package was used for these calculations. The level of significance chosen was 0.05 for all determinations.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Mesna alone did not induce MN in PCEs. Cyclophosphamide (CP) caused an induction of increased MN frequencies. The same effects were observed irrespective of the staining and scoring method used.
- Ratio of PCE/NCE (for Micronucleus assay): The PCE/NCE ratio was the highest in Mesna-treated rats and similar to vehicle control (physiological saline group of rats). The ratio PCEs/NCEs was lowered by CP compared to controls or Mesna-treated rats, indicating bone marrow toxicity. This effect was statistically significant.
- Statistical evaluation: Two-way analysis of variance showed no interaction between treatment and staining method which means that, as to the effect of treatments, similar results were obtained with both methods.

Any other information on results incl. tables

Co-administration of Mesna

Induction of Micronuclei

CP caused an induction of increased MN frequencies which was not significantly affected by co-administration of Mesna.

Ratio of PCE/NCE

Co-administration of Mesna with CP did not significantly improve the PCE/NCE ratio compared to CP alone.

Influence of staining procedure on the outcomes of MN in PCEs and PCE/NCE ratio

Slightly lower MN frequencies in controls and slightly higher CP-induced MN frequencies by Hoechst-Pyronin staining were observed. The PCE/NCE ratio was consistently higher in Hoechst-Pyronin-stained slides than in May-Grünwald-Giemsa-stained slides. Two-way analysis of variance showed no interaction between treatment and staining method which means that, as to the effect of treatments, similar results were obtained with both methods.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Mesna did not induce Micronuclei in polychromatic erythrocytes in rat bone marrow. The ratio PCE/NCE was not affected indicating no bone marrow toxicity. Mesna was not genotoxic in this study.
Data is taken from a study of high quality on a suitable Read-Across substance. Hence, data can be considered to be reliable to draw conclusions within a WoE approach on the potential of SPS to induce micronuclei or in general chromosome mutations in mammalian cells. In vitro, a second suitable RA substance, MPS, did not induce micronuclei, providing hence consistent results. The negative results of this assay are furthermore consistent with the ones of Mesna, which did not induce chromosome aberrations, SCEs or cell cycle modulations in mammalian cells in the supporting study.

Executive summary:

The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vivo in rats by analysing micronuclei in bone marrow ((Laehdetie et al., 1990). The study was aimed to elucidate whether or not Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and/or by suppressing the mutagenicity of the active metabolites of CP.Adult male Wistar rats received cyclophosphamide (30 mg/kg bw) and/or Mesna (15 mg/kg bw) intraperitoneally. Both chemicals were dissolved in physiological saline. With regard to Mesna, physiological saline was the negative control and cyclophosphamide was the positive control. The animals were killed 30 hours after the injections and bone marrows were removed and analysed for presence of micronuclei. Micronuclei were scored in May-Grünwald-Giemsa-stained and fluorescent preparations. The proportion of polychromatic erythrocytes (PCE) was determined per 1000 normochromatic erythrocytes (NCE), and the frequency of Micronuclei was counted in 1000 NCEs per animal. Ratios PCE/NCEs were calculated per dose group and analysed for significance statistically.

Mesna alone did not induce micronuclei in polychromatic erythrocytes in rat bone marrow, while CP caused an induction of increased MN frequencies. The ratio PCE/NCE was not affected in Mesna-treated rats and was similar to vehicle control, indicating no bone marrow toxicity. Opposite to this, the ratio PCEs/NCEs was lowered by CP compared to controls or Mesna-treated rats. This effect was statistically significant. If Mesna was co-administered with CP, the frequency of bone marrow micronuclei was not diminished. Co-administration of Mesna with CP did not significantly improve the PCE/NCE ratio compared to CP alone. May-Grünwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results.

Mesna is a suitable Read-Across substance for SPS as Dimesna and Mesna could be considered a metabolite of each other, which allows the conclusion that the same also applies for SPS and MPS, and their respective alkyl side chains differ only in one –CH₂- group, so it can reasonably concluded that underlying effects for a possible induction of micronuclei or chromosome mutations in general are comparable, which is in detail outlined in the read-across justification.