Disodium 3,3'-dithiobis[propanesulphonate]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction: Fertility study, rats, Sprague Dawley, female, iv injection of 2x 200 mg/kg bw 1 week apart pre-mating, Mesna: NOAEL ≥ 200 mg/kg bw, no effects on any of the reproductive outcomes
Toxicity to reproduction: Fertility study, rabbits, New Zealand White, male, iv injection of 10 weekly doses of 6, 9 or 12 mg/kg bw, 10 weeks postobservation, Mesna: NOAEL ≥ 9 mg/kg bw, no treatment related changes observed in all parameters observed, i.e. reproductive organ weights, gross histopathological lesions, testicular histological examinations, sperm characteristics

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no studies via oral, dermal or inhalative application route available. However, there are two Klimisch 2 Database entries on a suitable Read-Across substance (Mesna) available, and several studies on other reproductive toxicity studies and supporting developmental toxicity studies which are suitable to provide additional, relevant information to fully cover the required endpoint under REACH according to the requirements in Annex XIII.

The first iv study on rats, in which males were exposed 9 weeks prior/during mating and females 2 weeks prior/during mating and 7 days after conception revealed a TDLo of 26880 mg/kg bw (total dose), based on preimplantation mortality. No other effects on dams, embryos or foetuses were noted. Hence, with a total exposure time of 21 days, the total dose, TDLo of 26880 mg/kg bw, corresponds to 1280 mg/kg bw/day. Although no other effects on the dams were reported, this may nevertheless be related to maternal toxicity or, if not, the magnitude of the dosage, which is additionally due to the iv application 100% bioavailable, is way beyond any physiologically relevant exposure and does not support the conclusion that SPS is directly toxic to reproduction.

For the second study in rabbits, similar considerations apply, the dams were administered daily 13 doses in total, resulting in a total dose 7800 mg/kg bw. This corresponds to a LOAEL of 600 mg/kg bw/day being totally bioavailable, and may similarly be considered as not directly toxic to reproduction.

 

As it will be shown below, there are two additional fertility studies on either female rats and male rabbits, in which it was shown that Mesna and hence SPS does not have any effect on either female reproductive outcome nor male reproductive parameters. The known reproductive toxicants cisplatin (known to cause damage to ovaries in rats and humans) resp. ifosfamide did induce reprotoxic effects, clearly indicating that the test method is appropriate to detect possible reprotoxic effects. Hence, this information supports the conclusion that SPS does not need to be regarded as reproductive toxicant.

 

Also, there are five studies / database entries on developmental toxicity available, showing that even at very high doses (i.a. above limit test concentrations) Mesna and hence SPS is not a developmental toxicant of induces foetal toxicity, furthermore supporting the conclusion of non-classification.

 

In summary, the tonnage-driven data requirement under REACH are met, there is no indication that the results are not relevant for human risk assessment, as both rats and rabbits were used and the applied dosages even with no observed effects exceed in magnitudes the possibly expectable exposure in humans. So, no further testing is required to safely conclude that SPS does not need to be classified as reproductive toxicant.

Short description of key information:
There is no information on oral, dermal or inhalative route available, but this section can be covered with iv application data:
Toxicity to reproduction: Reproductive toxicity study, rats, iv application during mating (9 weeks (m), 2 weeks(f)), 7 days after conception (f), Mesna: TDLo = 26880 mg/kg bw (total dose), preimplantation mortality, no other effects on dams or fetuses
Toxicity to reproduction: (Reproductive)/Developmental toxicity screening study, rabbits, iv application during days 6 -18 of gestation, Mesna : TDLo = 7800 mg/kg bw (total dose), preimplantation mortality, no other effects on dams or fetuses

Effects on developmental toxicity

Description of key information

Developmental Toxicity: Teratogenicity study, rats, (Sprague-Dawley), single iv administration of 5 / 30 mg/kg bw on gestation day 13, Mesna: NOAEL = 30 mg/kg bw, no effects on malformations, dead or resorbed foetuses, foetal body weights
Developmental Toxicity: Embryotoxicity study, up to 2000 mg/kg, oral, gestation day 8 – 15, rats, or up to 1000 mg/kg, gestation day 7 – 17, rabbits, Mesna: No embryotoxic effects
Developmental Toxicity: Embryo/fetotoxicity study, rats, iv application of 8800 mg/kg (total dose) on gestation day 7-17, Mesna: TDLo = 8800 mg/kg bw (total dose), based on reduced body weight gain of newborn rats, reduced viability index, one stunted foetus. No death of foetuses was observed.
Developmental Toxicity: Embryo/fetotoxicity study, rats, iv application of 4400 mg/kg (total dose) on gestation day 7-17, Mesna: TDLo = 4400 mg/kg bw (total dose), based on behavioural effects on newborns and effects on the musculoskeletal system.
Developmental Toxicity: Embryo/fetotoxicity study, rats, iv application of 10400 mg/kg (total dose) on gestation day 7-17, Mesna: TDLo = 10400 mg/kg bw (total dose), based on one stunted foetus. No death of foetuses was observed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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

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:

no guideline followed

Principles of method if other than guideline:

Pregnant Sprague-Dawley rats were divided into nine treatment groups. Individual groups were administered 0.9% NaCl or cyclophosphamide (10 or 15 mg/kg) alone or in combination with MESNA at one of two doses (5 or 30 mg/kg), or MESNA alone on Day 13 of gestation.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc. (St. Constant, Quebec).
- Weight at study initiation: 225-250 g
- Housing: McIntyra Animal Center (McGill University, Montreal, Quebec)
- Diet (e.g. ad libitum): ad libitum (Purina Rat chow)
- Water (e.g. ad libitum): ad libitum

Route of administration:

intravenous

Vehicle:

physiological saline

Details on exposure:

MESNA or 0.9% NaCl was injected intravenously via jugular vein immediately followed by an ip dose of cyclophosphamide or 0.9% NaCl.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

single injection

Frequency of treatment:

single treatment on day 13 of gestation

Duration of test:

20 Days

Remarks:

Doses / Concentrations:
5 and 30 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

7 (65 animals were divided into nine treatment groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the MESNA doses of 5 and 30 mg/ kg were chosen to achieve ratios of MESNA to cyclophosphamide on a milligram per kilogram basis of I:3 (5 m/kg MESNA, 15 mg/kg cyclophosphamide; optimal for protection against urotoxicity) and 3: 1 (30 mg/kg MESNA, 10 mg/kg cyclophosphamide).

Maternal examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: not reported

Ovaries and uterine content:

No data

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Statistics:

Teratogenicity data were analysed with the fetus as the experimental unit by x² test with Yate's correction for discontinuity. The fetal weight data were analysed by one-way analysis of variance. The level of significance for all tests was taken as p<=0.05 unless stated otherwise. These procedures are described by Snedecor and Cochran (1967).

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:not examined

Remarks on result:

not measured/tested

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Mesna did not induce teratogenic effects in pups of rats until 30 mg/kg bw.

Executive summary:

The teratogenicity potential of MESNA could be evaluated from the results of a teratogenicity study conducted with cyclophosphamide known anti-cancer drug (Slott and Hales, 1986). The study was designed to determine whether the teratogenic effects of cyclophosphamide and its metabolites could be prevented by MESNA. On day 13 of gestation, pregnant Spague Dawley rats were administered MESNA intravenously at one of two dose levels (5 and 30 mg/kg bw) in combination with cyclophosphamide or alone. Vehicle control group included animals treated with 0.9% NaCl. Rats were killed on day 20 of gestation and fetuses were examined for gross external malformations, fetal body weight, and skeletal defects. The administration of MESNA alone had no significant effects on the incidence of malformations compared to vehicle control (2/62 malformed fetuses in 5 mg/kg bw group and 0/62 malformed fetuses in 30 mg/kg bw dose group). The administartion of MESNA had no sigificant effects on the incidence of dead or resorbed fetuses (similar to vehicle control). No effects of MESNA treatments were observed on fetal body weights. No skeletal malformations were recorded for MESNA treatment groups. MESNA significantly decreased a certain number of fetal malformations induced by cyclophosphamide administration confirming its protective effects against the teratogenic effects of cyclophosphamide.

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. This is in detail outlined in the read-across justification. Hence, it can be concluded that the derived NOAEL of 30 mg/kg is applicable for SPS, too.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

other: rats and rabbits

Quality of whole database:

There are in total 5 studies (iv and oral application) available, consistently indicating that mesna and hence SPS is not a developmental toxicant. Hence, the results can be considered as reliable, and no further developmental toxicity study is required, hence, the tonnage-driven data requirements are fully met. So the database is of high quality and the information is suitable to support the toxicity to reproduction endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a teratogenicity study in rats, there were no dead or resorbed foetuses or reduced foetal body weights observed after a single iv injection of 30 mg/kg bw on GD 13, indicating that this dose would not lead to reproductive toxicity in terms of foetal toxicity. Similarly, even very high doses of 2000 mg/kg bw (rats, GD 8-15) or 1000 mg/kg bw (rabbits, GD 7-17) does not result in any foetotoxic effects.

There are also three database entries of a embryo/fetotoxicity study in rats with iv applications of Mesna on GD 7-17. The following total doses, denoted as TDLo, were given, which can be converted into LOAELs based on different effects noted:

- TDLo = 4400 mg/kg bw (total dose), ≙ LOAEL = 400 mg/kg bw/d, based on behavioural effects on newborns and effects on the musculoskeletal system.

- TDLo = 8800 mg/kg bw (total dose), ≙ LOAEL = 800 mg/kg bw/d, based on reduced body weight gain of newborn rats, reduced viability index, one stunted foetus. No death of foetuses was observed.

- TDLo = 10400 mg/kg bw (total dose), ≙ LOAEL = 945 mg/kg bw/d, based on one stunted foetus. No death of foetuses was observed.

Also here, it is clearly indicated that Mesna, and hence SPS, does not lead to reproductive toxicity in terms of foetal toxicity.

Also, with regard to developmental toxicity / teratogenicity, the given data indicate that Mesna and hence SPS is not a developmental toxicant.

Justification for selection of Effect on developmental toxicity: via oral route:
only oral study available

Toxicity to reproduction: other studies

Additional information

The two available fertility studies assessed with Klimisch 2 on either female rats of male rabbits indicate consistently that Mesna and hence SPS does not have any impacts on female reproductive performance, e.g. number of resorptions, total number of corpora lutea, fecundity index, postimplantation losses etc., or male reproductive parameters, i.e. reproductive organ weights, gross histopathological lesion induction, testicular histological examinations, or sperm characteristics.

The present studies were not conducted under the currently recommended OECD guidelines, but performed in a scientifically reasonable manner with regard to the parameters in question. This endpoint is not required under REACH, so here are no data gaps, the given information is suitable to conclude in a WoE approach with the above-mentioned iv reproductive toxicity studies that SPS is not a reproductive toxicant.

Justification for classification or non-classification

The available reproductive toxicity study on rats and the (reproductive)/developmental toxicity screening study in rabbits both reveal a very high LOAEL, which is not relevant for human exposure. At this dosage, the reproduction-related effects are likely due to overall toxicity, and in summary not indicative for reproductive toxicity in humans.

 

All 5 available studies / database entries clearly reveal not fetotoxic / developmentally toxic effect such as malformations or other, even at doses up to 2000 mg/kg, both in rats and rabbits. Hence, Mesna and so SPS does not need to be regarded as a developmental toxicant.

 

The two available fertility studies on either female rats of male rabbits indicate consistently that Mesna and hence SPS does not have any impacts on female reproductive performance, e.g. number of resorptions, total number of corpora lutea, fecundity index, postimplantation losses etc., or male reproductive parameters, i.e. reproductive organ weights, gross histopathological lesion induction, testicular histological examinations, or sperm characteristics. Consequently, there is no indication given that SPS should be considered as reproductive toxicant.

 

So, in summary, there is no need to classify disodium 3,3'-dithiobis[propanesulphonate] (SPS) as reproductive / developmental toxicant.