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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented publication which meets basic scientific principles

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 specified
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

Constituent 1
Reference substance name:
Mesna
EC Number:
243-285-9
EC Name:
Mesna
Cas Number:
19767-45-4
IUPAC Name:
sodium 2-sulfanylethanesulfonate
Test material form:
other: solution
Details on test material:
- Name of test material (as cited in study report): MESNA (Asta Werke, Bielefeld, F.R.G.)
- Molecular formula (if other than submission substance): C2H5NaO3S2
- Molecular weight (if other than submission substance): 164.18
- Smiles notation (if other than submission substance): C(S)CCS(=O)(=O)O{-}.[Na]{+}
- InChl (if other than submission substance): 1S/C2H6O3S2.Na/c3-7(4,5)2-1-6;/h6H,1-2H2,(H,3,4,5);/q;+1/p-1
- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: organosulphur compound
- Physical state: pulver (Sigma Aldrich)
- Analytical purity: 98%

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 (migrated information): 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.
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.