Tosyl chloride

Endpoint summary

Additional information

Bacterial mutagenicity:

There are two studies available evaluating bacterial reverse mutation:

 

Tosyl chloride was evaluated bacterial mutagenicity pre-GLP and according to a method similar to OECD 471 (plate incorporation).Salmonella typhimuriumTA 1535, TA 1537, TA 98, TA 1538 and TA 100 andSaccharomyces cerevisiaewere exposed to 1.0, 10.0, 100.0, 500.0 and 1000.0 microgram/plate. The negative control was the solvent DMSO. The positive control depended on the strain. The test was performed with replication.

Results: Tosyl chloride exhibited toxicity with all the strains except D4 at 500 and 1000 µg/plate. The results of the tests conducted on the compound in the presence and absence of a metabolic activation system were all negative.

 

An additional study available on Tosyl chloride is presented in the OECD SIDS dossier on 4-methylbenzenesulfonyl chloride. In this study Tosyl chloride was evaluated in aSalmonella typhimurium(TA1535, TA1537, TA98 and TA100) andE coli(WP2uvrA) reverse mutation assay according to OECD guideline 471 and 472 and in compliance with GLP.

A preliminary test was performed with and without metabolic activation (10% S9 v/v Aroclor 1254 induced rat liver S9-mix) on TA100 strain at concentrations of 0, 313, 625, 1250, 2500 and 5000 μg/plate. The main study was performed at dose levels at levels of 313, 625, 1,250, 2500 and 5000 μg/plate without S9, and at 79, 157, 313, 625, and 1250 μg/plate with S9. The negative control was the solvent DMSO. Per concentration three plates were evaluated. No independent repeat was performed.

Results: It is indicated that no toxicity was observed. The vehicle (dimethyl sulphoxide) 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 or without metabolic activation. A significant increase of revertants per plate compared to the control was seen with TA100 at 1250, 2500 and 5,000 μg/plate in the absence of S9, but not in the presence of metabolic activation. All other strains and concentrations showed no increase in number of revertants/plate.

 

Genotoxicity in mammalian systems:

Tosyl chloride was evaluated forin vivoclastogenic activity and/or disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in ICR mouse bone marrow. The assay design was based on OECD Guideline 474 and performed in compliance to GLP.

The test substance was dissolved in corn oil and dosed at 0, 40, 80 mg/kg bw via i.p. route to groups of 6 male mice for three consecutive days. A positive control group received 2 mg/kg of Mitomycin C by i.p. injection once. At least 2000 PCEs per animal were analyzed for the frequency of micronuclei.

Results: No deaths were observed in relation to the treatments. Signs of toxicity were observed in the two highest doe groups. In both the positive control and at the highest dose of 80 mg/kg bw a decrease in the PCE:NCE ratio was observed, indicative of cytotoxicity in bone marrow.

No statistically significant increase in micronucleus frequencies were seen in mice treated with up to 80 mg/kg of Tosyl chloride.

 

As the evaluation of mutagenicity in mammalian cells were lacking Tosyl chloride was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cellsin vitroaccording to OECD guideline 476 and in compliance to GLP.

In the selection of an appropriate solvent for this study,water/media was not considered suitable for Tosyl chloride in view of its rapid hydrolysis in aqueous media into p-tolenensulfonic acid and HCl, both strong acids. Water would therefore lead to the evaluation of p-tolenensulfonic acid rather than the best possible approach to evaluate the possible mutagenic properties of Tosyl chloride.

In above reported bacterial mutagenicity tests DMSO had been used as solvent. In a first attempt Tosyl chloride was dissolved in water-free DMSO. This resulted to an unexpected high toxicity and positive mutagenicity at the higher dose levels. Chemical experts indicated that Tosyl chloride as reactive chlorine actually reacted with DMSO with the forming of a reactive sulfoxomium ion (a substance that can be used in the preparation of epoxides). Subsequently a repeat was performed using ethanol as solvent, as in literature ethanol was indicated to be a possible solvent for PTSC. Although now no mutagenicity was observed, chemical experts indicated that Tosyl chloride most likely also reacted to the hydroxyl group of ethanol, similar as with hydrolysis in water, with the forming of a Toluenesulfonate-ester. After further consultation of chemical experts it was suggested to use NMP (polar aprotic solvent; i.e. a solvent that will dissolve many salts, but lack an acidic hydrogen) as a solvent which is expected to be more inert than DMSO.

 

In range finding studies NMP itself was shown to be cytotoxic at 1% (v/v) and therefore testing in the main study was done with NMP 0.1%.

In the main study the cells were treated with PTSC dissolved in NMP for 4 h in the presence and absence of S9-mix. Tested was up to the maximum achievable concentration on the basis of cytotoxicity. Both in the presence and absence of S9-mix​ ​the criterion is that the highest concentration for evaluation should result to a RTG value between 10% and 20% (or 1 concentration between 1% and 10% and 1 concentration between 20% and 30%).

The negative and positive controls resulted to the expected response and met acceptance criteria. The test is therefore considered valid.

 

The results of this study showed that in the absence of S9 (4-h exposure) at the concentrations 16, 19 and 22 µg/ml an increase in the average mutant frequency (MF) was observed of more than 88 or 126 mutants per 1,000,000 cells compared the control. At these concentrations, however, there is very high cytotoxicity (more than 90%) and these results were not considered relevant following the criteria indicated in the study plan. Exposures of 24-hours in the absence of S9-mix showed no increase at any of the dose levels tested, with the highest evaluated concentration of 15 µg/ml showing a very high toxicity with an RTG of 3%.

In the presence of S9 (4h exposure) at the concentrations of 27 and 30 µg/ml an increase in the average mutant frequency (MF) is observed of more than 88 or 126 mutants per 1,000,000 cells compared to the control. Although the highest concentration (30 µg/ml) results to a RTG value of 7% (93% cytotoxicity), the next lower concentration of 27 µg/ml shows a RTG 17%, and thus conforms to the cytotoxicity criteria for acceptability. In addition, the increase in mutations seems to be dose related. The study therefore concludes that Tosyl chloride is mutagenic in the presence of metabolic activation (S9 mix). Both in the absence and presence relatively more small than large colonies were observed. This observation may be indicative for a clastogenic mechanism.

 

These results do fit with the expected profile which indicatesthat SN2 nucleophilic substitution reactions on DNA are possible. These results indicate that the hydrolysis is not just fast enough to result to full hydrolysis of PTSC before it reaches the nucleus. Only in that respect it is unexpected to see a difference between the situation with and without metabolic activation. However, when looking at all data combined it becomes evident that the results from all tests are very comparable: There is no increase in mutations up to about 20 µg/ml (already too toxic in case of no S9-mix for 24-hours), but with increasing concentration both toxicity and mutations increase rapidly. The very fact that there is no difference in that respect between with and without S9 indicates that the effect is likely related to the substance itself and possibly and effect occurring early in the exposure to the substance when hydrolysis is not complete.

As additional comment it can be remarked thatthe rapid hydrolysis intop-Toluenesulfonic acidand HCl, both very strong acids, could possibly lead to an increase of pH. Unfortunately pH has not been measured after the end of the study, but visible check of the cultures did not indicate a shift in the colour of the medium, suggesting that the buffer capacity was sufficient.

 

 

Evaluation:

Tosyl chloride (PTSC) is a reactive substance and in the presence of water, PTSC is quickly hydrolysed to the acid p-toluenesulfonic acid (PTSAcid). PTSAcid has been evaluated non genotoxic (separately submitted IUCLID dossier). Therefore, although profiling of PTSC in the OECD QSAR Toolbox triggers an alert for DNA binding via nucleophilic (S_N2) substitution, in practice due to rapid hydrolysis it is not expected that PTSC will actually reach the DNA.

The available testing supports a general lack of genotoxicity, even when taken care that PTSC is kept as stable as possible to prevent hydrolysis before start of exposure. With such test design trying to expose cells as much as possible to the tosyl chloride rather than the hydrolysis product, there was only one result found positive in one of the repeats performed with S9 at the highest dose level possible before considered to be too toxic to be relevant (i.e. toxicity > 90%).

A complication in the testing for genotoxicity is that the substance can react with solvent. In the case of DMSO this leads to the formation of a reactive sulfoxomium. This could also explain the positive response in one of the available Ames studies (only in TA100 in the absence of S9). Testing with ethanol as solvent in the MLA test was completely negative, but as PTSC is also expected to react with ethanol with the forming of a Toluenesulfonate-ester, these results were also not considered valid for the assessment of genotoxic hazards.

In accordance to expectations, the availablein vitrodata show only a limited concern for possible genotoxicity, despite the fact that chemical profiling indicates a possible reactivity to DNA. Following hydrolyses to the non-genotoxic p-toluenesulfonic acid, PTSC is not expected to be taken up and become systemically available in the first place. This is confirmed in anin vivomicronucleus study showed negative results following adequate i.p. dosing and showing toxicity in bone marrow.

Justification for selection of genetic toxicity endpoint

No one specific study is selected. For each endpoint addressing bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a study is available.

Short description of key information:

Performed studies, covering in vitro bacterial mutagenicity, mammalian mutagenicity and clastogenicity by means of an in vivo micronucleus study, support the conclusion that although chemical profiling indicate a possible reactivity to DNA the rapid hydrolysis into the non-genotoxic p-toluenesulfonic acid precluding to exert its activity in the in vivo situation. The in vitro mutagenicity study (MLA) resulted to a positive response at the maximum possible concentration in view of toxicity in one of the repeats performed with S9. The in vivo micronucleus showed negative results, following i.p. dosing and showing toxicity in bone marrow.

A complication in the testing for genotoxicity is that the substance can react with solvent. In the case of DMSO this leads to the formation of a reactive sulfoxomium. This could also explain the positive response in one of the available Ames studies (only in TA100 in the absence of S9).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Performed studies, covering in vitro bacterial mutagenicity, mammalian mutagenicity and clastogenicity by means of an in vivo micronucleus study, support the conclusion that although chemical profiling indicate a possible reactivity to DNA the rapid hydrolysis into the non-genotoxic p-toluenesulfonic acid precluding to exert its activity in thein vivosituation. The in vitro mutagenicity study (MLA) resulted to only one positive response in one of the two repeats performed with S9 at the maximum concentration considered physiological relevant in view of toxicity. It was remarked in that study that both in the absence and presence relatively more small than large colonies were observed, indicative for a clastogenic mechanism.

The in vivo micronucleus showed negative results following i.p. dosing and showing toxicity in bone marrow.

Available data therefore does not justify classification for genotoxicity.