Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
1) Ames test, 1987, TA98, TA100, TA1535, TA1537, not mutagenic in TA100 and TA1537 (without S-9 mix) and TA98, TA100 and TA1537 (with S-9 mix), ambiguous result in strain TA1535 (3-fold increase in revertant frequency of TA1535 at a single dose in the absence of S-9 in Experiment 2, but such an increase did not occur in Experiment 1). 2) Toolbox prediction- Ames Test (negative); 3) Toolbox prediction- Ames Test (negative); 4) MNT, OECD 487(Bohnenberger, 2012); negative; 5) SCE, OECD 479 (CAS 19767-45-4) (Wilmer et al., 1986); negative; 6) MN in vivo (CAS 19767-45-4) (Laehdetie et al., 1990); negative.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-18 - 2012-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Guideline No. 487 "In vitro Mammalian Cell Micronucleus Test".
Deviations:
yes
Remarks:
slight modification of the expression phase and harvest time, which is not compromising the outcome of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor (23 years old) for the first experiment, from a 27 year-old female donor for Experiment IIA and from a 33 year-old female donor for Experiment IIB. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats
Test concentrations with justification for top dose:
Experiment I without S9-mix: 0, 13.6, 23.8, 41.7, 73.0, 127.8, 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Experiment IIA without S9-mix: 0, 13.6, 23.8, 41.7, 73.0, 127.8, 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Experiment I with S9-mix: 0, 13.6, 23.8, 41.7, 73.0, 127.8, 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Experiment IIA and B with S9-mix: 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water, the final concentration of deinonised water in the culture medium was 10 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent solvent controls (culture medium with 10 % deionised water) (local tap water deionised at Harlan CCR)).
Positive controls:
yes
Remarks:
Mitomycin C (2.0 µg/mL - Exp. 1), Demecolcin (150 ng/mL - Exp. 2), Cyclophosphamide (10 µg/mL - Exp. 1 and 2)
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Remarks:
Dilutions of stock solutions were prepared on the day of the experiment. Stability of Demecolcin, Mitomycin C and CPA in solution is unknown but a mutagenic response in the expected range is sufficient biological evidence of chemical stability.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 40 hours
- Exposure duration: 4 and 20 hours
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: at least 1000 binuecleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: other: To describe a cytotoxic effect the CBPI (cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis (7).

OTHER:
Evaluation criteria:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data.
b) The positive control substances should produce significant increases in the number of cells with micronuclei.

Evaluation of Results

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In absence and presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. In exp. IIA without S9 mix stat. sig. increases (0.8, 1.0 % micronucleated cells) were in range of historical controls.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on pH value was observed.
- Effects of osmolality: No relevant influence on osmolarity value was observed.
- Precipitation: No precipitation of the test item in the culture medium was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test item sodium 3-mercaptopropanesulphonate, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix in three independent experiments. The following study design was performed:

 

Without S9-Mix

With S9-Mix

 

Exp. I

Exp. IIA

Exp. I and IIB

Exposure period

 4 hrs

20 hrs

 4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Preparation interval

40 hrs

40 hrs

40 hrs

Total culture period

88 hrs

88 hrs

88 hrs

So, in Experiment I, the exposure period was 4 hours with and without S9 mix and in Experiment IIA, the exposure period was 20 hours without S9 mix. In Experiment IIB, the exposure period was 4 hours with S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostatis.

The highest applied concentration in this test on toxicity (2097.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (85 %) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and in accordance with OECD Guideline 487.

No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH value was observed.

No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis could be observed up to the highest applied concentration.

In the absence and the presence of S9 mix, no biologically relevant increase in the number of cells carying micronuclei was observed.

The micronucleus rates of the cells after treatment with the test item (0.25 - 1.00 % micronucleated cells) were close to the range of the solvent control values (0.20 - 0.65 % micronucleated cells) and within the range of the laboratory historical control data. However, in Experiment IIA in the absence of S9 mix statistically significant increases (0.8 and 1.0 % micronucleated cells) were observed after treatment with 223.6 and 2097.0 µg/mL. The values are in the range of the historical solvent control data (0.05 - 1.45 %micronucleated cells) and therefore biologically irrelevant.

Appropriate mutagens were used as positive controls. In both experiments, either Demecolcin (150.0 ng/mL), MMC (2.0 µg/mL) or CPA (10.0 µg/mL) were used as positive controls and showed distinct statistically significant increases in cells with micronuclei.

Conclusion:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, when tested up to the highest required concentration.

Therefore, sodium 3-mercaptopropanesulphonate is considered to benon-mutagenic in this in vitro micronucleustest, when tested up to the highest required concentration.

Table 2: Summary of results of the in vitro micronucleus test in human lymphocytes with sodium 3-mercaptopropanesulphonate

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Negative control

2.00

 

0.45

 

 

Solvent control1

1.98

 

0.25

 

 

Positive control2

1.74

25.7

11.90S

 

 

684.7

2.05

n.c.

0.45

 

 

1198.3

2.11

n.c.

0.25

 

 

2097.0

2.10

n.c.

0.35

Exposure period 20 hrs without S9 mix

IIA

40 hrs

Negative control

1.96

 

0.25

 

 

Solvent control1

1.90

 

0.25

 

 

Positive control3

1.51

46.9

3.00S

 

 

223.6

1.80

11.0

0.80S

 

 

1198.3

1.69

23.2

0.50

 

 

2097.0

1.67

25.2

1.00S

Exposure period 4 hrs with S9 mix

I

40 hrs

Negative control

1.82

 

0.30

 

 

Solvent control1

2.01

 

0.20

 

 

Positive control4

1.58

29.6

4.40S

 

 

684.7

1.92

9.7

0.30

 

 

1198.3

1.91

10.2

0.45

 

 

2097.0

1.90

11.6

0.35

IIB

40 hrs

Negative control

2.01

 

0.70

 

 

Solvent control1

1.91

 

0.65

 

 

Positive control4

1.71

29.7

6.00S

 

 

684.7

1.93

n.c.

1.00

 

 

1198.3

1.95

n.c.

0.85

 

 

2097.0

1.97

n.c.

0.65

*    For the positive control groups, the relative values are related to the negative controls; for the test item treatment groups the values are

related to the solvent controls

**     The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S       The number of micronucleated cells is statistically significantly higher than corresponding control values

1       Deionised water 10.0 % (v/v)

2           MMC                      2.0 µg/mL

3       Demecolcin     150.0 ng/mL

4           CPA                     10.0 µg/mL

Table 3: Cytotoxicity of sodium 3-mercaptopropanesulphonate to the cultures of human lymphocytes.

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

4 hrs

40 hrs

2.00

---

Solvent control

4 hrs

40 hrs

1.98

---

13.6

4 hrs

40 hrs

n.d.

n.d.

23.8

4 hrs

40 hrs

n.d.

n.d.

41.7

4 hrs

40 hrs

n.d.

n.d.

73.0

4 hrs

40 hrs

n.d.

n.d.

127.8

4 hrs

40 hrs

n.d.

n.d.

223.6

4 hrs

40 hrs

2.02

n.c.

391.3

4 hrs

40 hrs

2.06

n.c.

684.7

4 hrs

40 hrs

2.05

n.c.

1198.3

4 hrs

40 hrs

2.11

n.c.

2097.0

4 hrs

40 hrs

2.10

n.c.

With S9 mix

Negative control

4 hrs

40 hrs

1.82

---

Solvent control

4 hrs

40 hrs

2.01

---

13.6

4 hrs

40 hrs

n.d.

n.d.

23.8

4 hrs

40 hrs

n.d.

n.d.

41.7

4 hrs

40 hrs

n.d.

n.d.

73.0

4 hrs

40 hrs

n.d.

n.d.

127.8

4 hrs

40 hrs

n.d.

n.d.

223.6

4 hrs

40 hrs

1.94

7.0

391.3

4 hrs

40 hrs

1.87

13.9

684.7

4 hrs

40 hrs

1.92

9.7

1198.3

4 hrs

40 hrs

1.91

10.2

2097.0

4 hrs

40 hrs

1.90

11.6

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*     Mean value of two cultures
n.d. Not determined
n.c.   Not calculated as the CBPIwas equal or higher than solvent control value

Toxicity - Experiment IIA

In Experiment IIA the CBPI in two cultures (500 cells per culture) was determined.

Table 4: Cytotoxicity of sodium 3-mercaptopropanesulphonate to the cultures of human lymphocytes.

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

20 hrs

40 hrs

1.96

---

Solvent control

20 hrs

40 hrs

1.90

---

13.6

20 hrs

40 hrs

n.d.

n.d.

23.8

20 hrs

40 hrs

n.d.

n.d.

41.7

20 hrs

40 hrs

n.d.

n.d.

73.0

20 hrs

40 hrs

n.d.

n.d.

127.8

20 hrs

40 hrs

n.d.

n.d.

223.6

20 hrs

40 hrs

1.80

11.0

391.3

20 hrs

40 hrs

1.78

13.8

684.7

20 hrs

40 hrs

1.70

22.0

1198.3

20 hrs

40 hrs

1.69

23.2

2097.0

20 hrs

40 hrs

1.67

25.2

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*       Mean value of two cultures
n.d.   Not determined

Toxicity - Experiment IIB

In Experiment IIB the CBPI in two cultures (500 cells per culture) was determined.

Table 5: Cytotoxicity of sodium 3-mercaptopropanesulphonate to the cultures of human lymphocytes.

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

4 hrs

40 hrs

2.01

---

Solvent control

4 hrs

40 hrs

1.91

---

223.6

4 hrs

40 hrs

1.97

n.c.

391.3

4 hrs

40 hrs

1.94

n.c.

684.7

4 hrs

40 hrs

1.93

n.c.

1198.3

4 hrs

40 hrs

1.95

n.c.

2097.0

4 hrs

40 hrs

1.97

n.c.

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*     Mean value of two cultures
n.c. Not calculated as the CBPI was equal or higher than solvent control value



Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD Guideline 487 with deviations (expression phase and harvest time were slightly modified - these deviations are not considered to influence the outcome of the study) and is considered to be of the highest quality (reliability Klimisch 1). The vehicle water and the positive control substances fulfilled validity criteria of the test system. The positive controls mitomycin C, demecolcin and cyclophosphamide induced micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. None of the cultures treated with 3-mercaptopropanesulphonate in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of micronuclei. Based on this test, 3-mercaptopropanesulphonate is considered not to be non-mutagenic in human lymphocytes.
Executive summary:

The test item 3-mercaptopropanesulphonate, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro (Bohnenberger, 2012). Cultured human lymphcytes were used and the following study design was performed: experiment 1without S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 2a without S9 -mix (exposure period 20 hrs, Cytochalasin B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 1 and 2b with S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalasin B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours). In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenic damage on coded slides. The highest applied concentration in the pre-test on toxicity (2097.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (85 %) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In the absence and the presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. However, in experiment IIa in the absence of S9 mix statistically significant increases (0.8 and 1.0 % micronucleated cells) were observed after treatment with 223.6 and 2097.0 µg/mL. The values are clearly within the range of the historical control data (0.05 - 1.45 % micronucleated cells) and therefore regarded as biologically irrelevant. Appropriate mutagens were used as positive controls. The positive controls mitomycin C, demecolcin and cyclophosphamide induced statistically significant increases in cells with micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. .

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, sodium 3 -mercaptopropanesulphonate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in vitro - gene mutation:

1-propanesulfonic acid, 3-mercapto-, monosodium salt was assayed for mutation in four histidine requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium both in the absence and presence of metabolic activation by an Aroclor-1254 induced rat liver post-mitochondrial fraction (S-9) in two separate experiments (Kennelly, 1987). The study was performed according to the OECD Guideline 471 with deviations (only 4 Salmonella strains tested) and considered to be reliable with restrictions (reliability Klimisch 2). An initial toxicity range-finder experiment was carried out in TA100 only, using final concentrations of 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and positive control. No toxicity was observed, therefore 5000 µg/plate was chosen as the top dose for the mutation experiments. In the first mutation experiment each strain was treated with the test substance in a five-fold concentration series up to 5000 µg/plate. For the second experiment, the dose range was narrowed in order to investigate effects at higher concentrations i.e., 500, 1000, 1500, 3000 and 5000 µg/plate. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. So the study met the acceptance criteria and is considered to be valid. Treatment of strains TA100 and TA1537 in the absence of S-9, and strains TA98, TA100 and TA1537 in the presence of S-9 in Experiments 1 and 2, gave rise to numbers of revertant colonies that were similar to those observed on concurrent solvent control plates. In none of these cases was there a reproducible two-fold (TA98 and TA100) or three-fold (TA1537) increase in revertant numbers. TA98 in the absence of S-9 in Experiment 2 exhibited a significant increase (2.1-fold) in revertants at 1500 µg/plate, but a similar response was not seen in Experiment 1. A 3-fold increase in revertant frequency of TA1535 was observed at a single dose in the absence of S-9 in Experiment 2, but such an increase did not occur in Experiment 1. However small increases (at least 1.9 x background) in TA1535 revertants, which exceeded the historical control range, were observed after all treatments at 5000 µg/plate in the absence and presence of S-9 in both experiments. Data provided by the Sponsor indicated that the sample of Compound 14:MPS was only 90.6% pure. The possibility that the small increases in reversion frequency were attributable to a contaminant cannot therefore be ignored. It is concluded that although the results did not fully satisfy the requirements for a mutagenic response, there were sufficient indications of a positive effect in strain TA1535 to prevent the compound being considered non-mutagenic in this assay. It is possible that the effect may have been the result of an impurity in the test sample.

The ambiguous findings in this study, including the fact that only 4 Salmonella strains were tested, was the reason to get information on the missing strains (E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 and/or S. typhimurium TA1535) from structurally similar chemicals.

The chemical 3-mercaptopropanesulfonic acid, sodium salt(CAS 17636 -10 -1) was evaluated by the OECD QSAR Toolbox (v.3.0) for its gene mutation potential in bacterial strains. The prediction (based on read-across) was based on the experimental values of chemicals assigned into the category. The target chemical was profiled as "Thiols "Acute toxicity" by the "US EPA New Chemical Categories". Thiols in general are known to be involved in the generation of reactive sulfur species through formation of free radicals (disulfide-S-monooxide or disulfide-S-dioxide etc.) leading to DNA adducts. This mechanism of action is relevant for the investigated endpoint. Therefore, primarily a search for chemicals with the same profiling result was performed. In the created category, there were chemicals which tested strains were defined and chemicals with undefined test strains. Therefore two predictions have been run. The first category (with defined test strains) was refined: the chemicals have been eliminated which mechanism of binding to DNA was other than "Radicals, generation of ROS; Thiols". The chemicals with other chemical elements in their structures have also been considered dissimilar and removed from the domain. The remaining chemicals in the category possess all thiol as the functional group and have a very similar mechanistic and endpoint specific profiling (similar reactivity to biomolecules). The second category was not refined as all chemicals were considered to be similar and suitable for read-across. The read-across chemicals in both categories do not contain a sulfonic group in their structure. The sulfonic group, however, is not associated with a high reactivity to biomolecules. All category members were negative in the Ames Test.

Genetic toxicity in vitro - chromosome aberration:

The test item 3-mercaptopropanesulphonate, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytesin vitro (Bohnenberger, 2012). Cultured human lymphocytes were used and the following study design was performed: experiment 1without S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalasin B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 2a without S9 -mix (exposure period 20 hrs, Cytochalasin B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 1 and 2b with S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalasin B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours). In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenic damage on coded slides. The highest applied concentration in the pre-test on toxicity (2097.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (85 %) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In the absence and the presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. However, in experiment IIa in the absence of S9 mix statistically significant increases (0.8 and 1.0 % micronucleated cells) were observed after treatment with 223.6 and 2097.0 µg/mL. The values are clearly within the range of the historical control data (0.05 - 1.45 % micronucleated cells) and therefore regarded as biologically irrelevant. Appropriate mutagens were used as positive controls. The positive controls mitomycin C, demecolcin and cyclophosphamide induced statistically significant increases in cells with micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. .

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, sodium 3 -mercaptopropanesulphonate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

SCE Test conducted with structural analogue mesna (CAS 19767 -45 -4)

Mesna was studied for its efficacy to attenuate the cytogenetic damage and cytotoxicity in cultured human lymphocytes exposed to cyclophosphamide (CP) and its reactive intermediates acrolein (AC), phosphoramide mustard (PAM), and diethyl-4'-hydroperoxycyclophosphamide (DEHP-CP) (Wilmer et a., 1986). Purified mononuclear leukocytes were stimulated with concanavalin A and exposed to CP at the concentrations of 0.5-2.0 mM without an exogenous activation system, or AC at the concentrationss of 0.001-40.0 µM, or PAM at concentrations of 0.0014-27.1 µM, or DEHP-CP at concentrations of 0.1-100 µM in the presence or absence of mesna at the concentration of 1, 5, or 10 mM. Negative controls were mesna alone and medium (untreated) cultures which were included in each experiment. The frequency of sister chromatide exchange (SCE), chromosome aberrations and cell cycle kinetics were measured.

CP, AC, DEHP-CP and PAM induced significant concentration-related increases in the SCE frequency. PAM was also clastogenic. Mesna alone did not induce SCE or perturb cell cyle kinetics. There were no significant increases in the number of chromosome aberrations compared to medium controls. Mesna added to CP-(and its reactive metabolites) treated cultures reduced significantly the SCE frequency and protected almost completely against cell lethality. Mesna decreased the percentage of cells with chromosome aberrations induced by PAM. The results of this study suggest that the anticarcinogenicity of mesna correlates with its ability to attenuate the cytogenetic damage and cytotoxicity induced by CP metabolites.

Genetic toxicity in vivo - Micronucleus Test conducted with structural analogue mesna (CAS 19767-45-4)

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 is the negative control and cyclophosphamide is 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.


Justification for selection of genetic toxicity endpoint
GLP guideline study conducted with mammalian cells.

Justification for classification or non-classification

Based on the negative result of the Chromosome Aberration Test conducted with the target substance MPS and negative result in in vivo Micronucleus Test conducted with the nearest analogue mesna, sodium 3 -mercaptopropanesulfonate does not need to be classified and labelled as genotoxic according to Regulation 1272/2008/EC (CLP).