Disodium 3,3'-dithiobis[propanesulphonate]

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

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:

other: Acceptable well-documented publication which meets basic scientific principles.

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.

Objective of study:

excretion

other: protective efficacy against ifosfamide induced urotoxicity

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mesna was tested for its uroprotective efficacy in 10 (per dose level) Sprague Dawley rats treated with 68 mg/kg ifosfamide that is known to induce haemorrhagic cystitis. Mesna was administered by i. v. administration 15 min before the injection of ifosfamide at dose levels of 6.81, 10.0, 14.7 and 21.5 mg/kg bw. The uroprotective efficacy of Mesna was evaluated 24 hours after the administration of ifosfamide. The rats were killed and the urinary bladder were evaluated (inflammation, bleeding and weight). The pharmakokinetic behaviour of mesna was also studied.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Asta-Werke, Bielefeld, and Mus Rattus AG, Brunnthal
- Weight at study initiation: 250 g
- Housing: standard conditions
- Diet (e.g. ad libitum): ad libitum (altromin ® 1324, no deprivation)
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS: not reported

Route of administration:

intravenous

Vehicle:

physiological saline

Details on exposure:

No details reported

Duration and frequency of treatment / exposure:

single treatment

Remarks:

Doses / Concentrations:
Screening dose: 100 mg/kg bw;
Further doses: 6.81, 10.0, 14.7 and 21.5 mg/kg bw

No. of animals per sex per dose / concentration:

10

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

Ifosfamide (68 mg/kg bw)

Details on study design:

- Dose selection rationale: screening dose 100 mg/kg, raised or lowered in steps with a factor of 2.15.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (excretion)
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: not reported in details (most likely: every hour)

Statistics:

No data

Type:

excretion

Results:

After i.v. administration of 21.5 mg/kg to the rat, about 40% of the administered dose is excreted with the urine as the sulfhydryl compound within the first hour.

Details on excretion:

Within the first 3 hour about 50% of the dose is excreted as the sulfhydryl compound and a further 30% of the dose as the disulphide. Rapid renal excretion of sulfhydryl groups is also observed after oral administration of mesna to rats. Similar findings have also been made on the dog. The behaviour of dimesna after intravenous and after oral administration to rats and dogs is very similar to that of mesna. After i.v. administration of 42.4 mg/kg to rats, 28% of the dose was excreted with the urine as the free sulfhydryl compound within 3 hours.

The severity of the inflammation of the bladder after i.v. administration of ifosfamide was dose-dependent. An ifosfamide dose of 68 mg/kg always induced an approximately 2- fold increase in the wet weight of the bladder and a damage score of about 2. Some of the rats also showed bladder haemorrhages. The scoring system used takes into account two parameters: firstly, increased capillary permeability (which is demonstrable objectively by the extravasal occurrence of intravenously injected trypan blue) and secondly, the increase in the weight of the bladder, which can also be assessed macroscopically as a swelling of the bladder. In some selected groups the bladder damage and the extent of bladder protection were also investigated histologically. The uroprotective effect of mesna was dose-dependent. It was reflected in a reduced increase in the bladder weight and reduced extravasation of trypan blue, and it was also demonstrable histologically. In the case of mesna, the lowest dose ensuring reliable uroprotection was determined. The results of these investigations of the uroprotective efficacy of MPS and mesna (as well as it dimer dimesna) are summarized in the following table:

Table 1.  Uroprotective action of mercapto-alkane sulfonates and analogues
					Assessment of urinary bladder
Compound	Structural formula	Dose (mg/kg)		Animals (n)	Inflamm. (x/n)	Bleeding	Weight (mg)		Score (0-3)
						x/n	Mean+S.E.
Untreated controls		—		105	0	0	81.0±12.0		0
Ifosfamide		68.1	i.v.	100	100	63	165.0±35.0		2.3
Mesna	HS-CH2-CH2 -SO3Na	6.81	i.v.	10	6	1	97.3±22.6		1.5
		10.0	i.v.	10	2	0	88.8±5.4		0.5
		14.7	i.v.	10	0	0	77.5±10.9		0.3
		21.5	i.v.	10	0	0	72.6±10.3		0
Dimesna	S-CH2CH2-S03Na S-CH2CH2-S03Na	21.5	i.v.	10	6	3	137.5±29.1		1.4
		31.6	i.v.	10	3	0	101.6±12.4		0.3
		46.4	i.v.	10	1	0	86.6±12.4		0.1
		68.1	i.v.	10	0	0	77.5±10.9		0
Asta 7100	HS-(CH2)3-SO3Na	21.5	i.v.	5	5	0	138.4±26.5		2.0
		68.1	i.v.	5	3	0	80.4±7.4		0.5
		215.0	i.v.	5	1	0	77.0±10.1		0.1

The longer chain homologue of mesna, Asta 7100 (= MPS), was also effective in protection of urinary bladder against ifosfamide induced urotoxicity.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Mesna protected completely ifosfamide induced urotoxicity in rats. Mesna was rapidly excreted in the urine. Data is taken from an acceptable well documented publication which meets basic scientific principles on a suitable Read-Across substance. Hence, data can be considered to be reliable within an WoE approach to conclude that no potential to bioaccumulate is given for Mesna or its category members.

Executive summary:

Mesna was tested for its uroprotective efficacy in 10 (per dose level) Sprague Dawley rats treated with 68 mg/kg ifosfamide that is known to induce haemorrhagic cystitis (Brock et al., 1981). Mesna was administered by i. v. administration 15 min before the injection of ifosfamide at dose levels of 6.81, 10.0, 14.7 and 21.5 mg/kg bw. The uroprotective efficacy of Mesna was evaluated 24 hours after the administration of ifosfamide. The rats were killed and the urinary bladder were evaluated (inflammation, bleeding and weight). The pharmakokinetic behaviour of mesna was also studied.

The severity of the inflammation of the bladder after i.v. administration of ifosfamide was dose-dependent. An ifosfamide dose of 68 mg/kg always induced an approximately 2- fold increase in the wet weight of the bladder and a damage score of about 2. Some of the rats also showed bladder haemorrhages. The scoring system used takes into account two parameters: firstly, increased capillary permeability (which is demonstrable objectively by the extravasal occurrence of intravenously injected trypan blue) and secondly, the increase in the weight of the bladder, which can also be assessed macroscopically as a swelling of the bladder. In some selected groups the bladder damage and the extent of bladder protection were also investigated histologically. The uroprotective effect of mesna was dose-dependent. It was reflected in a reduced increase in the bladder weight and reduced extravasation of trypan blue, and it was also demonstrable histologically. In the case of mesna, the lowest dose ensuring reliable uroprotection was determined (10 mg/kg bw). The longer chain homologue of mesna, MPS, the proposed metabolite of SPS, was also effective in protection of urinary bladder against ifosfamide induced urotoxicity. Regrading excretion of mesna, after i.v. administration of 21.5 mg/kg to the rat, about 40% of the administered dose is excreted with the urine as the sulfhydryl compound within the first hour. Within the first 3 hour about 50% of the dose is excreted as the sulfhydryl compound and a further 30% of the dose as the disulfide. Rapid renal excretion of sulfhydryl groups is also observed after oral administration of mesna to rats. Similar findings have also been made on the dog. The behaviour of dimesna after intravenous and after oral administration to rats and dogs is very similar to that of mesna. After i.v. administration of 42.4 mg/kg to rats, 28% of the dose was excreted with the urine as the free sulfhydryl compound within 3 hours.

Mesna is a suitable Read-Across substance for SPS asDimesna 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 e.g. absorption, distribution patterns, or excretion from organ systems and body in total are comparable, which is in detail outlined in the read-across justification.

Reference 2

Endpoint:

basic toxicokinetics in vivo

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.

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The pharmacokinetics and metabolism of mesna and its disulfide form (dimesna) have been investigated in the intact rat and in several in vitro systems, including isolated perfused organs, freshly isolated cells, and subcellular fractions; the mechanism of reduction of dimesna to form the pharmacologically active thiol mesna has been further studied with purified enzyme preparations.

GLP compliance:

no

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Weight at study initiation: 200 to 300 g
- Fasting period before study: no
- Housing: in stainless steel cages
- Individual metabolism cages: yes (for studies on the course of elimination of mesna or dimesna in the urine)
- Diet (e.g. ad libitum): pelleted rat food (ad libitum)
- Water (e.g. ad libitum): tap water (ad libitum)

Route of administration:

other: i.p., i.v. and p.o.

Vehicle:

not specified

Details on exposure:

Not applicable

Duration and frequency of treatment / exposure:

single application

Remarks:

Doses / Concentrations:
100 mg/kg bw (all routes)

No. of animals per sex per dose / concentration:

20 (excretion)

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

None

Details on study design:

Not applicable

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum or other tissues, cage washes, bile

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, tissues, cage washes, bile
- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC)

Statistics:

Not reported

Type:

absorption

Results:

After p.o. administration, mesna and dimesna are both absorbed from the intestine, and dimesna undergoes reduction to mesna during intestinal absorption.

Type:

excretion

Results:

Independently of the route of administration,mesna or dimesna were excreted almost completely within 6 hr in the rat as free thiols.

Type:

distribution

Results:

Mesna and Dimesna pass unchanged through the hepatic vasculature, are not taken up into liver cells, and are not excreted in bile; uptake of Mesna by small intestines and kidney was observed.

Details on absorption:

Also, after p.o. administration, both mesna and dimesna were readily absorbed, as demonstrated by significant elevation of disulphide concentration in plasma; free thiols were hardly or not at all observed. Mesna seemed to be absorbed somewhat better (or earlier) during the intestinal passage than dimesna was. Due to a prolonged absorption phase, the plasma concentration of reducible disulfides increased only slowly and reached a plateau maximum between 1 and 3 hr after administration.
After i.v. injection of mesna (100 mg/kg) into rats, there was an increase in the concentration of free thiols in plasma, which disappeared within minutes; a concomitant increase in the level of reducible disulphides was observed during a much longer time period. The plasma disappearance rate of the sum of administered thiols and immediately oxidized disulphides after i.v. injection of mesna was almost identical to the rate of plasma elimination of disulphides after administration of dimesna.
After i.p. administration of mesna or dimesna, there were no apparent differences in the low plasma concentration of free thiols and the high concentration of reducible disulphides. The rate of absorption from the peritoneal cavity appeared to be equally fast for both compounds, and peak plasma concentrations were reached within the first 10 min after administration. Furthermore, the slope of the disappearance curve for plasma reducible disulfides, after i.p. administration of either mesna or dimesna, was similar to the slope of the plasma disappearance curve observed after i.v. administration.

Details on distribution in tissues:

Mesna and dimesna were readily absorbed from the gastrointestinal tract after p.o. administration to rats in vivo. To further substantiate this finding, an isolated, inverted intestinal segment was incubated with 1 mM [14C]dimesna in vitro, and the amount of radioactivity was measured in the medium and in the lumen contents. After 60 min of incubation, the concentration of radiolabel in the lumen contents was almost twice that originally present in the surrounding medium (191 ± 28%). It is of interest to note that, in spite of the fact that there were no measurable free thiols in the medium during the entire experiment, the lumen contents of the closed segment contained a slowly increasing amount of thiol groups, amounting to 30 to 60% of the accumulated radioactivity. During control incubations in the absence of dimesna, no luminal thiol accumulation occurred.
Uptake of dimesna was also observed in experiments with isolated cells from small intestine and kidney but not with hepatocytes. In order to investigate whether this uptake was coupled to membrane-bound -γ-glutamyltransferase activity, which is abundant in intestine and kidney but very low in liver in the rat, a series of experiments were performed with the potent γ -glutamyltransferase inhibitor, anthglutin, added to the cellular incubation at a final concentration of 2 mM. This resulted in almost complete inhibition of γ -glutamyltransferase activity without measurable effect on the rate of dimesna uptake in any of the cell types investigated.
When the isolated rat liver was perfused with [14C]dimesna, there was no detectable extraction of radiolabel during passage through the hepatic vascular bed, no excretion of dimesna or mesna in bile, and no accumulation of thiol or radiolabel in the liver tissue. Thus, it appears that the liver is totally inactive in the
whole-body handling of mesna and dimesna in the rat. When similar experiments were performed with the isolated perfused rat kidney, [14C]dimesna gradually disappeared from the recirculating perfusate but never appeared in urine when the perfusate concentration of dimesna was kept below 2 mM. On the other hand, whereas reactive mesna was excreted in the urine at high concentrations, it could be detected only in trace amounts in the perfusate leaving the kidneys. Compared to clearance data for exogenously added creatinine (1.6 ± 0.2 mL/min), the extraction of dimesna from the perfusate during kidney passage was somewhat lower (1.0 ± 0.1 mL/min). This observation indicates that, in addition to glomerular filtration, dimesna undergoes reabsorption from the tubular fluid, a process which is compatible with the aforementioned intracellular reduction mechanism. Furthermore, it seems that after reduction most of the thiol formed is reexcreted back into the tubular lumen, whereas any disulphide exceeding the cellular reduction capacity is transported across the basal part of the tubular plasma membrane to reenter the peritubular vessels.

Details on excretion:

Independently of the route of administration of mesna or dimesna, the renal elimination rate corresponded quite well to the disappearance of reducible disulphides from peripheral plasma. The metabolic output is very low in untreated animals. A spontaneous urinary excretion rate (n = 50) of 0.58 ±0.44 µmol/kg/hr for free thiols and 2.46 ± 1.25 µmol/kg/hr for thiols after reduction of disulphides was found. These values would correspond to an excretion rate for mesna of 0.1 or 0.4 mg/kg/hr, respectively. Renal excretion of mesna or dimesna started immediately after i.v. injection, whereas it was slightly delayed after i.p. administration, and it showed a plateau maximum from 1 to 3 hr after p.o. administration. In all instances, the dose of mesna or dimesna administered was excreted
almost completely within 6 hr in the rat. Moreover, in all experiments, significant amounts (40 to 70%) of the total dose of mesna or dimesna given appeared as free thiol in the urine. This was in contrast to the rapid disappearance of free thiol in plasma and suggested a complex system of oxidations and reductions during the whole-body turnover of mesna and dimesna.

Metabolites identified:

yes

Details on metabolites:

After i.v. injection, mesna rapidly disappeared from plasma and was replaced by dimesna. It is oxidised by a metal dependent reaction. In the kidney, dimesna is filtered through the glomeruli and subsequently reabsorbed, whereupon reduction to the pharmacologically active thiol form occurs in the renal tubular epithelium, and the thiol is then reexcreted into the tubular lumen. Reduction of dimesna to mesna occurs in intestinal and renal epithelial cells by a mechanism involving the cytosolic enzymes thiol transferase and glutathione reductase.

Localisation of oxidation reactions and metabolism

Prompted by the observation that, after i.v. injection, mesna rapidly disappeared from plasma and was replaced by dimesna, a series of experiments were performed to investigate the nature and localization of the oxidation reaction. When mesna was incubated in vitro under various conditions, the rate of spontaneous oxidation in plasma-free medium (pH 7.4, carbogen gassing) was relatively slow . Addition of washed human or murine erythrocytes to the incubation did not change this oxidation rate, whereas, in the presence of whole blood or plasma, the rate of oxidation of mesna was much enhanced. Addition of metal-chelating agents, notably EDTA, strongly inhibited the rate of mesna oxidation.

There was no apparent formation of mesna from dimesna during incubation of the disulphide in buffer, plasma, or urine. In contrast, incubation of dimesna with cells, isolated from either small intestine or kidney, was associated with reduction of the disulphide and accumulation of increasing concentrations of free thiol in the medium. On the other hand, incubation of dimesna with isolated hepatocytes did not result in any increased level of mesna in the medium, suggesting that the reduction of dimesna to mesna is mediated by an intracellular mechanism and occurs only in cells which can take up the disulphide. This hypothesis received further support from the results of experiments with dialised cytosolic fraction of a kidney homogenate.

Incubation of dimesna with the dialyzed cytosolic fraction of a kidney homogenate in the presence of added NADPH and GSH resulted in rapid conversion of the disulfide to the thiol; other renal subcellular fractions tested were inactive in this respect. Moreover, when the dialyzed cytosolic fraction was replaced by purified preparations of thiol transferase and GSH reductase, the reduction of dimesna to mesna was shown to proceed at a similar rate. The involvement of thiol transferase in the reduction of dimesna was further substantiated by similar experiments with the cytosolic fraction of a liver homogenate, which was also found to catalyse the GSH- and NADPH-dependent reduction of dimesna to mesna. Thus, it appears that the difference in capacity to reduce dimesna to mesna, observed with isolated cells from the various tissues, was related to differences in the ability to take up the disulphide rather than to lack of the enzymatic mechanism involved in dimesna reduction.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Mesna is rapidly oxidized to dimesna in peripheral blood after i.v. administration. Mesna and dimesna are readily absorbed via the oral route. Dimesna is taken up by the small intestines and the kidneys. In the kidney, it is reabsorbed, reduced to active thiol and then reexcreted into the lumen. Data is taken from an acceptable well documented publication which meets basic scientific principles on a suitable Read-Across substance. Hence, data can be considered to be reliable within an WoE approach to conclude that no potential to bioaccumulate is given for SPS.

Executive summary:

The pharmacokinetics and the metabolism of mesna and dimesna have been investigated in the intact rat and in several in vitro systems including isolated perfused organs, freshly isolated cells, subcellular fractions (Ormstad et al., 1983). The mechanism of reduction of dimesna to form the pharmacologically active thiol mesna has been further studied with purified enzyme preparations.

After p.o. administration, mesna and dimesna are both absorbed from the intestine, and dimesna undergoes reduction to mesna during intestinal absorption. When present in plasma, mesna is rapidly oxidized to dimesna by a metal-dependent reaction. Mesna and dimesna pass unchanged through the hepatic vasculature, are not taken up into liver cells, and are not excreted in bile. In the kidney, dimesna is filtered through the glomeruli and subsequently reabsorbed, whereupon reduction to the pharmacologically active thiol form occurs in the renal tubular epithelium, and the thiol is then reexcreted into the tubular lumen. Reduction of dimesna to mesna occurs in intestinal and renal epithelial cells by a mechanism involving the cytosolic enzymes thiol transferase and glutathione reductase. Thus, the formation of the pharmacologically active thiol form from dimesna is associated with the consumption of equimolar concentrations of reduced glutathione.

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 e.g. absorption, distribution patterns, or excretion from organ systems and body in total are comparable, which is in detail outlined in the read-across justification.

Reference 3

Endpoint:

basic toxicokinetics in vivo

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:

other: Acceptable well documented publication which meets basic scientific principles.

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.

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

other: binding to plasma proteins

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mesna was studied in rats for its toxicokinetic behaviour. Serum half-life, binding to proteins, distribution to organs and excretion have been investigated.

GLP compliance:

no

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 549 ± 126 g

Route of administration:

intravenous

Vehicle:

physiological saline

Duration and frequency of treatment / exposure:

single injection

Remarks:

Doses / Concentrations:
50 mg/5 µCi in saline

No. of animals per sex per dose / concentration:

9

Control animals:

no

Positive control reference chemical:

no

Details on study design:

no data

Type:

absorption

Results:

Mesna and dimesna do not enter most cells. Mesna is very well absorbed from the GI tract but dimesna is less readily absorbed; i.v. mesna and dimesna are pharmacokinetically undistinguishable.

Type:

distribution

Results:

mesna is predominantly present in plasma and extracellular fluids but it is taken up by kidney cells.

Type:

other: binding to plasma proteins

Results:

small proportion (9.7%) of circulating mesna/dimesna is bound to plasma proteins, including albumin and immunoglobulins.

Type:

excretion

Results:

The high water solubility of mesna and dimesna facilitate their rapid clearance from plasma by the kidney (within 24 h approx. 77% of the administered dose appeared in the urine).

Details on absorption:

Both mesna and dimesna are too hydrophilic to cross lipid biological membranes passively and therefore they do not enter most cells.
Surprisingly, in view of its high polarity, mesna is very well absorbed from the gastrointestinal tract following oral dosing. It has a high apparent bioavailability, it is well tolerated by the oral route, and therefore this route of administration is potentially very useful as part of cancer chemotherapy regimens.

Details on distribution in tissues:

The calculated volume of distribution for mesna in the rat is 0.3 L/kg, which supports the hypothesis that mesna is predominantly present in plasma and extracellular fluids. It is, however, possible that membrane carrier systems (perhaps thiol amino acid carriers) might permit uptake of mesna or dimesna by specific cells. Indeed, this possibly explains why mesna or dimesna appear to enter kidney cells.

Details on excretion:

The high water solubility of mesna and dimesna facilitate their rapid clearance from plasma by the kidney. This is reflected in their short plasma half-lives. The proportion of mesna to dimesna excreted into the urine is very much greater than the corresponding proportion in plasma. This is important, since it is mesna (rather than dimesna) which is responsible for detoxification of oxazaphosphorine metabolites. The reason for the free thiol being excreted in urine is that
enzymes in kidney cells are able to reduce dimesna to mesna. This reaction appears to involve concomitant oxidation of GSH. Mesna forms a mixed disulphide with cysteine, and one would expect that reduction of this disulphide, in a manner analogous to the postulated reduction of dimesna by kidney cells prior to urinary elimination, would result in excretion of cysteine in the urine. Indeed, the excretion of cysteine in urine following mesna administration has been demonstrated in both animals and man. There are other possible mechanisms which might account for the enhanced elimination of cysteine during mesna administration. Interaction of mesna with cystine is known to release cysteine with concomitant formation of mesna-cysteine disulphide. If this occurs in the circulatory system, enhanced cysteine elimination might result. Another possible mechanism, strictly speaking an in vitro artefact, results in the release of cysteine from
cystine following its reaction with mesna in the urinary bladder. This again would result in enhanced levels of cysteine in the urine.

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 4 h (following administration of [35S]-mesna

Test no.:

#2

Toxicokinetic parameters:

half-life 2nd: 17 min (following administration of [14C]-mesna

Metabolites identified:

yes

Details on metabolites:

On entering the blood stream, mesna is almost immediately oxidized to its dimeric form, dimesna (dithio-bis-mercaptoethanesulphonate) by forming a disulphide bridge between two monomeric units. This reaction is possibly spontaneous, being catalysed by transition metals present in the circulatory system, although it is possible that enzymic catalysis is involved. Mixed disulphides with endogenous thiols are also formed, for example cysteine-mesna disulphide. Dimesna is chemically less reactive than mesna and so is less likely to react with metabolites of anticancer drugs.

Binding to plasma proteins

A small proportion (10%) of circulating mesna/dimesna is bound to plasma proteins, including albumin and immunoglobulins. This has been studied in animals using [14C]-mesna. The binding mechanism is thought to involve both disulphide formation between mesna and cysteinyl thiol groups on the protein molecule and/or electrostatic interactions between positive regions of the protein molecule and the negatively charged sulphonate group of mesna or dimesna.

Plasma half-life

Pharmacokinetic studies using radiolabelled mesna have produced apparently conflicting results relating to plasma half-life values. If radioactivity is determined in whole blood following i.v. administration of [35S]-mesna and the half-life calculated, a value of some 4 h is obtained, while measurement of plasma radioactivity in a similar experiment using [14C]-mesna gives a half-life of some 17 min. This large discrepancy might be attributable to sequestration of mesna/dimesna by erythrocytes. Erythrocytes contain high levels of free thiols (e.g. GSH), and formation of disulphides with mesna is therefore very likely. This increased half-life would be related to the slow release of sequestrated mesna from the erythrocytes

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Mesna is well absorbed from GI tract. In blood stream, it is rapidly oxidized to dimesna. Its plasma half-life is 17 min. Mesna binds to plasma proteins (albumin and immunoglobulins). It is taken up by kidney cells and rapidly eliminated in the urine.
Data is taken from an acceptable well documented publication which meets basic scientific principles on a suitable Read-Across substance. Hence, data can be considered to be reliable within an WoE approach to conclude that no potential to bioaccumulate is given for SPS.

Executive summary:

In the review, the role of mesna as an uroprotective drug in anticancer therapy is presented. Mesna acts as a scavenger of acrolein and chloroacetaldehyde, reactive metabolites of oxazaphosphorines (cyclophosphamide, ifosfamide), preventing bladder toxicity during chemotherapy. The free thiol group of mesna can react with an electrophilic centre of the reactive compounds (epoxides, chlorinated hydrocarbons, free radicals etc.) forming a thioether, facilitating its excretion. Mesna is very well absorbed from GI tract following oral dosing. Mesna does not enter most cells due to its hydrophilicity and therewith does not reduce cytotoxicity of anticancer drugs in tumor cells. It is predominantly present in plasma and extracellular fluids but is taken up by kidney cells. On entering the blood stream, mesna is almost immediately oxidized to its dimeric form dimesna by forming a disulphide bridge. Dimesna is chemically less reactive than mesna and so is less likely to react with oxazaphosphorine metabolites. A small proportion (9.7%) of circulating mesna/dimesna is bound to plasma proteins, including albumin and immunoglobulins. The half-life of 17 min and 4 hours were calculated using [14C]-mesna and [35S]-mesna in radioactivity measurements in experimental animals, respectively. Mesna can be sequestred by erythrocytes, increasing its half-life. Mesna is rapidly eliminated in the urine. 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 e.g. absorption, distribution patterns, or excretion from organ systems and body in total are comparable, which is in detail outlined in the read-across justification.

Description of key information

Toxicokinetics - Serum half-life, binding to proteins, distribution, excretion: in vivo, male Wistar rats, ip & oral application, read-across from Mesna, CAS 19767-45-4: Mesna can react with an electrophilic centre of epoxides, chlorinated hydrocarbons, free radicals etc. forming a thioether, facilitating its excretion. Mesna is very well absorbed from GI tract, it is predominantly present in plasma and extracellular fluids, where it is almost immediately oxidized to its dimeric form dimesna by forming a disulphide bridge. 9.7% of circulating mesna/dimesna is bound to plasma proteins. Mesna is rapidly eliminated in the urine. Mesna, Dimesna and hence SPS has no potential for bioaccumulation.
Toxicokinetics – Excretion: in vivo, Sprague-Dawley rats m/f, iv injection, read-across from Mesna, CAS 19767-45-4: About 40% of 21.5 mg/kg is excreted with the urine as sulfhydryl compound within 1h. Within the first 3 hours about 50% of the dose is excreted as the sulfhydryl compound and a further 30% of the dose as the disulphide. Mesna, Dimesna and hence SPS has no potential for bioaccumulation.
Toxicokinetics – pharmacokinetics, metabolism: In vivo & in vitro, male Sprague-Dawley rats, ip, iv, oral administration, perfused kidney, metabolic incubation, read-across from Mesna, CAS 19767-45-4: Mesna is rapidly oxidized to dimesna in peripheral blood after i.v. administration. Mesna and dimesna are readily absorbed via the oral route. Dimesna is taken up by the small intestines and the kidneys. In the kidney, it is reabsorbed, reduced to active thiol and then reexcreted into the lumen. Mesna, Dimesna and hence SPS has no potential for bioaccumulation.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

50

Additional information

There are three Klimisch 2 literature sources available on the read-across substances Mesna resp. Dimesna, clearly indicating that SPS has no potential for bioaccumulation. There are no species-specific absorption mechanisms known and no data indicates that those are existing. Hence, every result and conclusion derived from the available studies in rats can be transferred to human risk assessment. Also, there are no indications given that an additional CH2-group has a relevant impact on the toxicokinetic behaviour, so that data from Mesna / Dimesna can be directly transferred to MPS (1-Propanesulfonic acid, 3-mercapto-, monosodium salt) / SPS.

 

Generally, Mesna and Dimesna could be considered a metabolite of each other. There are two known types of Metabolism of Mesna and Dimesna, i.e. enzymatic and non-enzymatic.

Early studies of Mesna’s in vivo fate reported complete spontaneous metal-catalyzed oxidation of Mesna within the circulation to its dimer, Dimesna.Recent studies of iv Dimesna pharmacokinetics show a dose-dependent increase in Mesna exposure of 6% to 8% of Dimesna AUC; intracellular Dimesna concentrations have been hypothesized to be less than 1% of the Mesna concentration.

In addition to non-enzymatic (i.e. chemical) thiol exchange, thioredoxin and glutaredoxin systems of the thioredoxin family of thiol-disulfide oxidoreductases may also be involved in the reduction of Dimesna and similarly the reduction of SPS. The reduction of Dimesna to the chemoprotectively active drug Mesna appears to arise from the preferential uptake of the drug in the kidneys, where Dimesna would undergo intracellular conversion into Mesna. In vitro, both liver and kidney lysates can reduce Dimesna to sulfhydryl Mesna by GSH dependent mechanisms. For further details, please refer to the extensive read-across justification. In summary, it is obvious that Dimesna is reduced to Mesna, and similarly SPS to MPS.

Either as conjugate or as highly soluble salt, the excretion can be, based on physico-chemical parameters, expected to be rather fast. This is shown by the available testing data, giving a consistent result. In combination with the negative LogPow, it can be safely concluded that SPS has no potential for bioaccumulation. This information is relevant for further risk assessment, as absorption of the compound is given to a rather high extent.

 

Concrete absorption rates are not given in any of the available data sources. However, based on the available information, they can be estimated.

As known from an oral study, within the first 3 hours after application of 21.5 mg/kg, about 50% of the dose is excreted as the sulfhydryl compound, i.e. Mesna, and a further 30% of the dose as the disulphide (Dimesna). Although SPS is the dimer of MPS, and has hence a nearly double molecular weight, it has still a rather moderate size (i.e. 354 g/mol) making it favourable for absorption. Also, water solubility (600 g/L resp. 1000 g/L (estimated)) and logPow (-3.68 resp. -2.94) are comparable between the dimer and monomer. This data is indicative for a similar absorption behaviour to MPS. As it is known that in total 80% of MPS is reexcreted after 3 hours, it is very likely that the substance is absorbed nearly completely, leading to the estimation of the absorption rate of 100%.

Information on dermal or inhalative absorption is not available. However, the lack of testing data does not trigger the necessity for additional testing as data can be, due to animal welfare, sufficiently reliable estimated from the available data, taking into account i.a. physico-chemical data and the recommendations from ECHA’s guidance R.7C “Endpoint specific guidance”.

 

With regard to inhalative absorption, according to ECHA’s guidance, moderate log P values (between -1 and 4) are favourable for absorption directly across the respiratory tract epithelium by passive diffusion. The determined LogPow is -3.68 at 23°C, lying clearly below that value and so hindering diffusion. Further, vapours of very hydrophilic substances may be retained within the mucus, whereas low water solubility enhances penetration to the lower respiratory tract. With a water solubility of 600 g/L at 25°C, absorption here is also hindered. Nevertheless absorption cannot be excluded, leading to the estimation of a precautionary absorption rate of 50% via inhalation.

 

With regard to dermal absorption, the guidance states: “If water solubility is above 10,000 mg/L and the log P value below 0 the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Dermal uptake for these substances will be low.“ Also, „For substances with log P values <0, poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. Values <–1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low.“ With a water solubility = 600 g/L at 25°C and LogPow = -3.68 at 23°C, the dermal absorption can be practically neglected. The relevant boundary value for low absorption is 10%, which is consequently set as the estimated dermal absorption rate for SPS. The conclusion of very diminished absorption is supported by the fact that an oral LD50 could be determined as 3720 mg/kg, whereas at a dose of 7500 mg/kg no signs of toxicity could be seen after dermal application.

 

In summary, all relevant information for the assessment of the toxicokinetic behaviour of SPS could be retrieved from the already available data of an acceptable quality, the dta requirements under REACH are met, and no additional testing is required.