2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethane-1-sulfonyl chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Study according to OECD 471 (1997): Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535, TA1537, concentration range 31.6 µg to 5000 µg/plate, with and without metabolic activation, TA-2 tested up to the cytotoxic concentration of 3160 µg TA-2/plate caused a pronounced concentration-related mutagenic effect in the test strains TA100 or TA98 in the plate incorporation test without or with metabolic activation, respectively, and in the test strains TA98, TA100, TA102 and TA1535 in the preincubation test carried out without and with metabolic activation at 1000 and/or 316 µg/plate.

- Study according to OECD 471 (1997), Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535, TA1537, concentration range 31.6 µg to 5000 µg/plate, with and without metabolic activation, vehicle N,N-dimethylformamide to prevent confounding effects from DMSO, negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The  potential  of  TA-2  to  induce  gene  mutations  was  examined  in  5  Salmonella typhimurium  strains  TA98,  TA100,  TA102,  TA1535  and  TA1537  in  two  independent experiments,  each  carried  out  without  and  with  metabolic  activation  (a  microsomal preparation  derived  from  Aroclor  1254-induced  rat  liver).  The  first  experiment  was carried out as a plate incorporation test and the second as a preincubation test.   TA-2 was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO was employed as the negative control.

In  the  plate  incorporation  test  and  in  the  preincubation  test  carried  out  without metabolic activation a pronounced concentration-related mutagenic effect (significant at p ≤  0.05)  and  at  least  2 -fold  increase  compared  to  the  negative  reference  item  was observed as follows:

-  Plate incorporation test without metabolic activation:

in test strain TA100 at a concentration of 1000 µg TA-2/plate;

-  Plate incorporation test with metabolic activation:

in test strain TA98 at a concentration of 1000 µg TA-2/plate;

-  Preincubation test without metabolic activation:

in test strains TA98, TA100, TA102 and TA1535 at a concentration of 1000 µg TA-2/plate and, in addition at 316 µg TA-2/plate in test strains TA98, TA100 and TA102;

-  Preincubation test with metabolic activation:

in test strains TA98, TA100, TA102 and TA1535 at concentrations of 316 and 1000 µg TA-2/plate.

All  criteria  for  a  positive  response  (a  concentration  (log  value)-related  effect  and  a 2-fold increase in the case of TA98, TA100 and TA1535 and a 1.5-fold increase in the case of TA102 in revertant colony numbers compared with control counts, significant at p ≤ 0.05) were met, in the experiments without metabolic activation (plate incorporation test and preincubation test).

No  mutagenic  effect  (no  increase  in  revertant  colony  numbers  as  compared  with control  counts)  was  observed  for  the  test  strain  TA1537  in  all  experiments  and, additionally, for the test strains TA98, TA102 and TA1535 in the plate incorporation test without metabolic activation and for the test strains TA100, TA102 and TA1535 in the plate incorporation test with metabolic activation.

The  positive  control  items  showed  a  significant  increase  in  the  number  of  revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

The test item TA-2 contains the reactive group sulfonyl chloride, and thus belongs to a group  of  substances  (carboxylic  acid  halides,  carbamoyl  halides,  thionyl  halides  and

sulfonyl halides), which have been described to produce positive results in the Ames test. However, positive results have generally been observed only when DMSO is used as  a  solvent,  whereas  negative  results  have  been  produced  when  other  organic solvents  are  used  (Amberg  et  al.,  2015).  A  possible  explanation  for  the  DMSO-dependent  activity  in  the  Ames test  involves  the  reaction  of these  substance groups with DMSO, resulting in the formation of the corresponding halodimethyl sulfide via the Pummerer rearrangement (March, 1992). In this context, Bolye (1966) had previously

reported  that  sulfonic  acid  chlorides  can  react  with  DMSO  to  form  chlorodimethyl sulfide, an alkylating agent expected to produce a positive response in the Ames test (Amberg etr al., 2015).

Therefore, the positive results observed  with TA-2 under the conditions of this study should  be  regarded  with  caution,  and  further  investigations  were made  to  elucidate whether the mutagenic effect may have been confounded by the possible reaction of TA-2 with the solvent DMSO.  

In a second experiment the  potential  of  TA-2  to  induce  gene  mutations  was  examined  in  5  Salmonella typhimurium  strains  TA98,  TA100,  TA102,  TA1535  and  TA1537  in  two  independent

experiments,  each  carried  out  without  and  with  metabolic  activation  (a  microsomal preparation  derived  from  Aroclor  1254-induced  rat  liver).  The  first  experiment  was carried out as a plate incorporation test and the second as a preincubation test.   TA-2 was completely dissolved in anhydrous N,N-dimethylformamide. The vehicle N,N-dimethylformamide was employed as the negative control.

No  increase  in  revertant  colony  numbers  as  compared  with  control  counts  was observed for TA-2, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains  in  two  independent  experiments  without  and  with  metabolic  activation, respectively (plate incorporation test and preincubation test).

The  positive  control  items  showed  a  significant  increase  in  the  number  of  revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Based on the weight of evidence from the two available studies, the substance is not considered to be mutagenic.

Justification for classification or non-classification

Testing of mutagenicity of TA-2 in the first study according to OECD guideline 471 showed a positive result. However, based on the results published in Amberg, A. et al. (2015) Do Carboxylic/Sulfonic Acid Halides Really Present a Mutagenic and Carcinogenic Risk as Impurities in Final Drug Products Organic Process Research and Development, 19(11):1495-1506, these mutagenic effects are considered to be based on the formation of reactive intermediates of the solvent and the test item. Thus, a second test according to OECD guideline 471 was conducted using Acetonitrile as solvent and revealed no mutagenic effect of the test substance. Hence, the present study was not used to evaluate the mutagenic potential of TA-2 due to confounding effects of DMSO. Thus, testing was repeated using N,N-dimethylformamide as vehicle. In the second test according to OECD guideline 471 no genotoxicity was observed.

Therefore, TA-2 is not classified as mutagenic according to Regulation (EC)No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).