Disodium 3,3'-dithiobis[propanesulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: Gene mutation (Bacterial reverse mutation assay): SPS, S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvr A: negative ±S9 (OECD 471, GLP)

In vitro: Gene mutation (Bacterial reverse mutation assay): MPS, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: negative ±S9, S. typhimurium TA 1535: positive (OECD 471, GLP)

In vitro: Chromosome aberration (Micronucleus test): MPS, human lymphocytes: negative ±S9 (OECD 473, GLP)

In vitro: Chromosome aberration, sister chromatid exchange, perturbance of cell cycle kinetics: Mesna, human lymphocytes: negative ±S9 (similar to OECD 479, OECD 473)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 July - 1 August 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Methods for Determination of Toxocity , Annex to Directive 92/69/EEC , Method B14 , Other effects - Mutagenicity : Samonella typhimurium - Reverse Mutation Assay

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: conforms to the guidelines for bacterial mutagenicity testing by the major Japanese Regulatory Authorities including MITI , MOL and MAFF

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(The Department of Health of the Government of the United Kingdom)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (Salmonella strains)
trp operon (Escherichia coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

incubated in the presence of a induced liver microsomal fraction (S9) prepared from rats

Test concentrations with justification for top dose:

First and second experiment : 50 , 150 , 500 , 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: the test material was soluble in water up to 50 mg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

See -Any other information on materials and methods incl. tables- for details

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates

NUMBER OF CELLS EVALUATED: 3 plates per dose level , in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Assessment for numbers of revertant colonies and examination for effects on the growth of the bacterial background lawn .


OTHER: In a range-finding study , five concentrations (50,150,500,1500 and 5000 µg/plate) were assayed in triplicate against each tester strain (with and without S9-mix) , using the direct plate incorporation method . Plates were incunated at 37°C for 48 h .

Evaluation criteria:

For a substance to be considered positive in this test system , it should have induced a dose-related and statistically significant increase in the revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels .
To be considered negative , the number of revertants at each dose level should be less than twofold to that of the vehicle control frequency .

Statistics:

A statistical analysis of the data was not required to determine the result of the test .

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable .

Remarks on result:

other: all strains/cell types tested

Table 1 : Range-finding study

		Mean number of revertant colonies per plate (average of 3 plates)
With or without S9 -Mix	Test substance concentration (µg/plate)	Base-pair substitution type			Frameshift type
		TA 100	TA 1535	WP2uvrA^-	TA 98	TA 1537
-	0	103	18	20	24	10
-	50	98	20	18	21	7
-	150	103	17	16	17	8
-	500	104	14	19	22	8
-	1500	95	19	17	23	9
-	5000	105	24	16	23	12
Positive controls , -S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (µg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3)	490	636	653	206	887
+	0	107	15	23	30	13
+	50	103	14	23	26	12
+	150	101	15	23	29	13
+	500	103	14	24	24	13
+	1500	101	18	21	28	13
+	5000	110	20	23	27	12
Positive controls , +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Mean No. of colonies)plate (average of 3)	1063	241	822	367	236

ENNG = N-ethyl-N´-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-1 -oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

Table 2 : Main study

		Mean number of revertant colonies per plate (average of 3 plates)
With or without S9 -Mix	Test substance concentration (µg/plate)	Base-pair substitution type			Frameshift type
		TA 100	TA 1535	WP2uvrA^-	TA 98	TA 1537
-	0	86	15	17	18	8
-	50	81	14	17	15	7
-	150	88	14	17	19	7
-	500	82	16	17	20	5
-	1500	91	16	17	18	6
-	5000	90	15	19	18	9
Positive controls , -S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (µg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3)	477	687	958	256	858
+	0	106	16	21	25	11
+	50	103	14	21	30	9
+	150	101	16	24	27	10
+	500	98	16	19	28	7
+	1500	111	17	21	30	8
+	5000	94	15	22	26	8
Positive controls , +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Mean No. of colonies)plate (average of 3)	1126	331	769	422	235

ENNG = N-ethyl-N´-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-1 -oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD TG471 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains , with any dose of the test material , either with or without metabolic activation . The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. lt also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

2012-04-18 - 2012-07-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

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.

Qualifier:

according to guideline

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. QA statement)

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 binucleate 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

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 control^1	1.98		0.25
		Positive control^2	1.74	25.7	11.90^S
		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 control^1	1.90		0.25
		Positive control^3	1.51	46.9	3.00^S
		223.6	1.80	11.0	0.80^S
		1198.3	1.69	23.2	0.50
		2097.0	1.67	25.2	1.00^S
Exposure period 4 hrs with S9 mix
I	40 hrs	Negative control	1.82		0.30
		Solvent control^1	2.01		0.20
		Positive control^4	1.58	29.6	4.40^S
		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 control^1	1.91		0.65
		Positive control^4	1.71	29.7	6.00^S
		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:
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.
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 vivo, a second suitable RA substance, Mesna, 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 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.

MPS is suitable as a read-across substance for SPS, as data derived from their structural analogues Dimesna and Mesna, which differonly in one –CH₂- group, show that these substances are turning into each other in biological systems, which is in detail outlined in the read-across justification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo: Chromosome aberration (Micronucleus test): Mesna: negative (similar to OECD 474, single ip injection of 15 mg/kg bw, mice)

Link to relevant study records

Reference

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.

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

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

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

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

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

There are several data available sufficiently covering all required and also additional endpoints, i.e. gene mutation, chromosome aberrations, DNA repair, in vitro and in vivo, and which are all assessed as at least Klimisch 1. All available data is consistently negative for genotoxic effects.

SPS was negative in an Ames Test, the RA substance MPS was tested negative in another Ames test, too. MPS was negative in an in vitro Micronucleus Test in human lymphocytes, as well as Mesna was negative in an in vivo Micronucleus Test in mice and an assay in human lymphocytes covering the endpoints Chromosome aberrations, sister chromatid exchange and cell cycle modulations. The latter two endpoints (negative) are indicative that no DNA damage repair has occurred, which may lead to mutations if not repaired error-free. No study on gene mutations in mammalian cells is available, however, this information is considered sufficient to conclude that testing would lead to a negative result. No hazard for humans could be identified, especially when taking into account the negative results of a carcinogenicity study in rats with Mesna. As carcinogenic effects, which are strongly related to gene mutations in mammalian cells, can be excluded, no additional genotoxicity testing is required. No indication is given that data on MPS or Mesna are not relevant for SPS, or that the obtained results are not relevant for humans.

Hence, the data base is of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from disodium 3,3'-dithiobis[propanesulphonate], no data gaps could be identified and no additional testing is required.

Justification for selection of genetic toxicity endpoint
Only in vivo study available. An in vivo study mimics the situation in humans more accurate than an in vitro study, and MESNA was identified as a suitable Read-across substance for SPS. However, selection has no influence on the key values for safety assessment as all available results on bacteria, mammalian cells, or in vitro and in vivo are negative.

Justification for classification or non-classification

All available test results for gene mutation, chromosome aberrations, DNA repair, in vitro and in vivo, and carcinogenicity, are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified.