Methyl methanesulphonate

Endpoint summary

Description of key information

Short-term toxicity to fish:

In a short term combine multigeneration reproductive and genotoxicity study, Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) in the concentration of 0, 2, 4 and 8 mg/L for 96 hours. Overall mortality, embryo and larval stages mortality were observed in 2, 4 and 8 mg/L dose. Similarly, Significantly decreased total length of fish, Premature gonads, delayed time point of the first spawning by 51 days and decreased number of eggs were observed in 2 mg/L dose. More prevalent female fish and Significantly more lethal and sublethal teratogenic effects in embryos and larvae were observed in 2 mg/L treated fish as compared to control. In addition, significantly elevated percentage of DNA in tail and liver cells from sexes as well as female gills and male gonads and significantly increased micronucleus frequencies in liver, gills and gonads of both male and female fish in 2 mg/L treated fish were observed as compared to control. Therefore, EC 50 was considered to be 2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hours.

Long-term toxicity to fish

In a long term Combine multigeneration reproductive and genotoxicity study, Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) in the concentration of 0, 0.2 and 0.4 mg/L for30 days. Overall mortality, embryo and larval stages mortality were observed in 0.2 doses. Similarly, significant increase in body weight of fish, Earlier spawn and increase egg productions were observed in 0.2 mg/L dose. Significantly more sublethal teratogenic effects in embryos and larvae were observed in 0.2 mg/L treated fish as compared to control. In addition, significantly elevated percentage of DNA in tail and liver cells from sexes as well as female gills and male gonads and significantly increased micronucleus frequencies in liver, gills and gonads of both male and female fish in 0.2 mg/L treated fish were observed as compared to control. Therefore, EC 50 was considered to be 0.2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hours. 

Short-term toxicity to aquatic invertebrates

Using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on aquatic invertebrates was predicted for methyl methanesulfonate(CAS:66-27-3). EC50 value was estimated to be 128.11 mg/l for Daphnia magna for 48 hrs duration. Based on the value,methyl methanesulfonate(CAS: 66-27-3) was considered to be non-toxic to aquatic invertebrates and can be considered to be not classified as per the CLP regulations.

Toxicity to aquatic algae and cyanobacteria

Short term toxicityChlamydomonas reinhardtiistudy was carried out for 60 mins(Eva Miadokova et. al; 2010). The test organism used for the study was unicellular green algaC. reinhardtii.Algal cells were treated with test chemical methyl methanesulfonate (0.1, 0.2 and 0.3%) for 60 mins in the dark and then plated on agar dishes supplemented with acetate. After 5 days of cultivation, survival was evaluated by a microscopic method that distinguished the algal cells having died due to cytotoxicity from the cells having died due to mutagenic (lethal mutations) effects of test chemical MMS. The test was performed under static conditions for 60 mins. For statistical analysis Student’s t-test was used. Based on mortality of the test organismChlamydomonas reinhardtii,the EC50 value was determined to be 1000 mg/l. Thus based on the EC50 value, it can be concluded that the substance Methyl methanesulfonate can be considered as non-toxic to aquatic organisms and thus can be considered to be not classified as per the criteria of CLP regulation.

Toxicity to microorganisms:

Based on the prediction done using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the six closest read across substances, toxicity on microorganism Tetrahymena pyriform is predicted for methyl methanesulfonate (66-27-3). Based on the growth rate inhibition, the IGC50 value was estimated to be 428.4 mg/l when methyl methanesulfonate (66-27-3) exposed to Tetrahymena pyriformis for 48hrs.

Additional information

Summarized result of toxicity of methyl methanesulfonate (MMS) (66-27-3) on the growth and other activity of fish, aquatic invertebrates, algae and cyanobacteria and microorganisms by considering the data for target as well as RA chemical which was selected on the basis of structural and functional similarity and log Kow as the primary descriptor are as follows:  

 

Short term toxicity to fish:

Based on the experimental data for the target chemical from various journals and publications, study have been reviewed to determine the toxic nature of target chemical methyl methanesulfonate (MMS) (66-27-3) on the growth, mortality and embryo development of fishes. The studies are as mentioned below:  

First experimental study (Ecotoxicology (2013) 22:825–837) suggest in a short term combine multigeneration reproductive and genotoxicity study, Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) in the concentration of 0, 2, 4 and 8 mg/L for 96 hours. Overall mortality, embryo and larval stages mortality were observed in 2, 4 and 8 mg/L dose. Similarly, significantly decreased total length of fish, premature gonads, delayed time point of the first spawning by 51 days and decreased number of eggs were observed in 2 mg/L dose. More prevalent female fish and significantly more lethal and sublethal teratogenic effects in embryos and larvae were observed in 2 mg/L treated fish as compared to control. In addition, significantly elevated percentage of DNA in tail and liver cells from sexes as well as female gills and male gonads and significantly increased micronucleus frequencies in liver, gills and gonads of both male and female fish in 2 mg/L treated fish were observed as compared to control. Therefore, EC 50 was considered to be 2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hours.

 

Whereas another authorative database (ECOTOX database by US EPA; 2017) indicate in Acute fish toxicity data the lowest observable effect concentration determine of test substance methyl methanesulfonate for exposure period of 24 hrs on the basis of Genetics effects. Test organism was used as Australoheros facetus (Chameleon Cichlid) in static freshwater with test temp. 18 deg.C, pH 8.2,Hardness 270.2 mg/L CaCO3 and alkalinity 160 mg/L CaCO3.In acute fish toxicity study the Lowest observable effect concentration (LOEC) on the basis of Genetics effect was observed to be 10 mg/l for exposure period of 24 hrs with significance level <0.05.

 

Similarly study from same data source suggest the lowest observable effect concentration of test substance methyl methanesulfonate for exposure period of 24 hrs. on the basis of Genetics damage effects. Test organism was used as Oreochromis niloticus (Nile Tilapia) in static freshwater with test temp. 25 deg.C and pH 7.0.In acute fish toxicity study the Lowest observable effect concentration (LOEC) on the basis of Genetics damage effect was observed to be 7.5 mg/l for exposure period of 24 hrs with significance level <0.0001.

 

In the fourth experimental study from peer reviewed journal (Environmental Toxicology and Chemistry, 1987) study was carried out and the effect was determine on the viability of fish for total exposure period of 2 hrs. The objective of this study was to determine the in vitro developmental toxicity of methyl methanesulfonate (MMS) on the (medaka) fish embryo culture systems as models for screening these developmental toxin. MMS were dissolved in 11 mM sodium citrate buffer (pH 6.0) immediately before use. The test solution was added directly to embryo rearing medium at final concentrations ranging from 0.75 mM to 120 mM (82 .59 mg/l to 13215 mg/l). Medaka (Oryzias latipes) was obtained from Carolina Biological, Burlington, North Carolina and were maintained in freshwater aquaria in our laboratory at a temperature range of 25 to 28°C. Fish fed Tetra-Min flakes and newly hatched brine shrimp larvae twice daily. In 60 mm x 15 mm Petri dishes Embryos were collected from female fish from two aquaria each containing 60 fish in a ratio of two males to three females. Eggs were pooled and then randomly distributed among experimental groups. All embryo experiments were conducted in isotonic rearing media at 20°C. Control embryos were exposed to buffer vehicle only. Treatment was initiated when embryos reached stage 20 (period of early organogenesis, approximately 66 h post fertilization at 20°C). After 2 h of exposure in the dark, embryos were extensively rinsed and transferred to Petri dishes containing fresh embryo rearing medium. All dishes were coded for blind observation. In addition, each experiment contained an uncoded control dish for comparative evaluation and photography. These values and their 95% confidence limits were calculated according to the modified probit analysis method. Based on the significant concentration -dependent decreases in viability, as expressed by the percentage of embryos that survived until hatching, the LC50 was determine to be 1740 mg/l after the exposure of chemical methyl methanesulfonate (MMS) with the medaka fish for 2 hrs. Based on the LC50 chemical was consider as nontoxic and not classified as per the CLP classification criteria.

 

Similarly fifth study was taken from the same from peer reviewed journal (Environmental Toxicology and Chemistry, 1987) in which effect of chemical on the embryonic development of Medaka (Oryzias latipes) was studied for the target chemical. The objective of this study was to determine the in vitro developmental toxicity of methyl methanesulfonate (MMS) on the (medaka) fish embryo culture systems and malformations. MMS were dissolved in 11 mM sodium citrate buffer (pH 6.0) immediately before use. The test solution was added directly to embryo rearing medium at final concentrations ranging from 0.75 mM to 120 mM (82 .59 mg/l to 13215 mg/l). Medaka (Oryzias latipes) was obtained from Carolina Biological, Burlington, North Carolina and were maintained in freshwater aquaria in our laboratory at a temperature range of 25 to 28°C. Fish fed Tetra-Min flakes and newly hatched brine shrimp larvae twice daily. In 60 mm x 15 mm Petri dishes Embryos were collected from female fish from two aquaria each containing 60 fish in a ratio of two males to three females. Eggs were pooled and then randomly distributed among experimental groups. All embryo experiments were conducted in isotonic rearing media at 20°C. Control embryos were exposed to buffer vehicle only. Treatment was initiated when embryos reached stage 20 (period of early organogenesis, approximately 66 h post fertilization at 20°C). After 2 h of exposure in the dark, embryos were extensively rinsed and transferred to Petri dishes containing fresh embryo rearing medium. All dishes were coded for blind observation. In addition, each experiment contained an uncoded control dish for comparative evaluation and photography. These values and their 95% confidence limits were calculated according to the modified probit analysis method. Based on the significant concentration -dependent increases in frequency of malformations the MC50 was 1376 mg/l after the exposure of chemical methyl methanesulfonate (MMS) with the medaka fish for 2 hrs. Based on the MC50 chemical was consider as nontoxic and not classified as per the CLP classification criteria.

 

Thus by considering all the studies. As the above three studies from Ecotoxicology (2013) 22:825–837) and secondary source ECTOX database suggest that the chemical was toxic as the effect concentration EC50 ranges from 2 mg/l to 10 mg/l which indicates that the chemical was consider as toxic and classified in aquatic chronic category 2. But the remaining 2 studies from peer reviewed journal (Environmental Toxicology and Chemistry, 1987) for the target chemical indicates and supports the nontoxic and not classified nature of target chemical methyl methanesulfonate (MMS). In the fourth and fifth studies, total exposure period of chemical with the test organism was very less (2 hrs.) and the study was not performed according to the standard OECD guidelines. Thus based on the overall criteria chemical was consider as toxic and can be consider to be classified in aquatic chronic category 2 as per the CLP classification criteria.

 

Long term toxicity to fish:

Two studies including experimental results of long term fish toxicity endpoint for test chemical methyl methanesulfonate (Cas no. 66-27-3) were summarized as followed:

First experimental study (Ecotoxicology (2013) 22:825–837)suggest a long term Combine multigeneration reproductive and genotoxicity study, Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) in the concentration of 0, 0.2 and 0.4 mg/L for 30 days. Overall mortality, embryo and larval stages mortality were observed in 0.2 doses. Similarly, significant increase in body weight of fish, earlier spawn and increase egg productions were observed in 0.2 mg/L dose. Significantly more sublethal teratogenic effects in embryos and larvae were observed in 0.2 mg/L treated fish as compared to control. In addition, significantly elevated percentage of DNA in tail and liver cells from sexes as well as female gills and male gonads and significantly increased micronucleus frequencies in liver, gills and gonads of both male and female fish in 0.2 mg/L treated fish were observed as compared to control. Therefore, EC 50 was considered to be 0.2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hours. 

 

Also supporting study (ECOTOX database by US EPA; 2017) indicate In long term fish toxicity study the Lowest observable effect concentration (LOEC) on the basis of Genetics damage effect was observed to be 0.11 mg/l for exposure period of 21 days. This effect concentration indicate that the test substance methyl methanesulfonate was toxic to fish in long exposure period and thus consider for the aquatic classification.

 

Thus based on the above available studies it is concluded that the test substance methyl methanesulfonate (Cas no. 66-27-3) consider to be toxic to fish in long exposure period and thus consider for the aquatic classification in chronic category 2 as per the CLP criteria based on the EC50 value of 0.2 mg/L.

 

Short term toxicity to invertebrates:

Based on the predicted data for the target chemical and experimental for read across chemicals study have been reviewed to determine the toxic nature of target chemical methyl methanesulfonate (MMS) (66-27-3) on the growth of aquatic invertebrates. The studies are as mentioned below: 

In the first weight of evidence study for the target chemical methyl methanesulfonate (MMS) (66-27-3) from OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on aquatic invertebrates was predicted for methyl methanesulfonate(CAS:66-27-3). EC50 value was estimated to be 128.11 mg/l for Daphnia magna for 48 hrs duration. Based on the value, methyl methanesulfonate (CAS: 66-27-3) was considered to be non-toxic to aquatic invertebrates and can be considered to be not classified as per the CLP regulations.

 

Similarly in the second predicted study for the chemical methyl methanesulfonate (MMS) (66-27-3) based on the prediction done by EPI suite, ECOSAR version 1.1, on the basis of similarity of structure to chemicals for which the aquatic toxicity has been previously measured by structure-activity relationships (SARs) program, the LC 50 value for short term toxicity to aquatic invertebrates was predicted. On the basis of this programe, the LC 50 value for short term toxicity to aquatic invertebrates was predicted to be 9800.8 mg/l for methyl methanesulfonate (66-27-3) in 48 hrs. Based on this value it can be concluded that the substance methyl methanesulfonate (66 -27 -3) is considered to be not toxic to aquatic environment and cannot be classified as per the criteria mentioned in CLP regulation.

 

Further predicted study was supported by the experimental study for the functionally similar read across chemical (121-57-3) used from the peer reviewed journal (Chemosphere, 1994). Toxicological profile of sulphanilic acid were study on the daphnia magna for 24 hrs. From the study the EC50 value for short term toxicity to aquatic invertebrates for Sulfanilic acid in 24 hrs was determined to be 109.13 mg/l. Based on the result, Sulfanilic acid (121-57-3) was considered as not toxic and not classified as per the CLP regulations.

 

Similarly study for the same read across chemical (121-57-3) from IUCLID dataset supports the nontoxic behavior of chemical. The short term toxicity study of sulphanilic acid to aquatic invertebrates was performed on Daphnia magna. Test was performed by static system. After the 24 hrs of exposure with chemical, the EC50 and EC100 value for Daphnia magna for Sulphanilic Acid (121-57-3) was determined to be 109.13 and 250 mg/l. Based on the value the sulphanilic Acid was considered to be non-toxic to aquatic invertebrate and can be considered to be not classified as per CLP regulations.

 

In the fifth weight of evidence study for another read across chemical 2,3-dibromopropan-1-ol (96-13-9) from ECOTOX database toxicity of chemical was studied. Short term toxicity to aquatic invertebrates was performed in 5 daphnia magna for 48 hrs at 289-395 range concentration. Age of 6-24 h old daphnia were used for experiment. The test was performed in 20 degree C and at pH of≥7. After the experiment, the EC 50 value for short term toxicity to aquatic invertebrates for 2,3-dibromopropan-1-ol (96-13-9) was determined to be 338 mg/l. Based on the value the chemical was considered to be non-toxic to aquatic invertebrates and can be considered to be not classified as per CLP regulations.

 

Thus based on the data obtain from various sources including predicted data for the target chemical (from OECD QSAR toolbox, EPIsuite) and for the read across chemical (from Chemosphere, 1994, IUCLID dataset and ECOTOX database, it can be concluded that the methyl methanesulfonate (MMS) (66-27-3) was nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

Various key and supporting study for the target chemical Methyl methanesulfonate (CAS no. 66 -27 -3) were reviewed for the toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In a key study, short term toxicity Chlamydomonas reinhardtii study was carried out for 60 mins (Eva Miadokova et. al; 2010). The test organism used for the study was unicellular green alga C. reinhardtii. Algal cells were treated with test chemical methyl methanesulfonate (CAS no. 66-27-3) (0.1, 0.2 and 0.3%) for 60 mins in the dark and then plated on agar dishes supplemented with acetate. After 5 days of cultivation, survival was evaluated by a microscopic method that distinguished the algal cells having died due to cytotoxicity from the cells having died due to mutagenic (lethal mutations) effects of test chemical MMS. The test was performed under static conditions for 60 mins. For statistical analysis Student’s t-test was used. Based on mortality of the test organism Chlamydomonas reinhardtii, the EC50 value was determined to be 1000 mg/l.

 

In a supporting study from peer reviewed journal (NEOPLASMA, 2006), short term toxicity Chlamydomonas reinhardtii study was carried out for 30 mins. The test organism used for the study was unicellular green alga C. reinhardtii recombination-repair-deficient strain uvs10.The life stage of the test organism was log growth phase. Algal cells were cultivated with a 12-h photoperiod in a liquid medium supplemented with acetate for 3–4 d (log growth phase) at 25°C and harvested by centrifugation for 7 min at 3,000 rpm. Subsequently, algal cell suspensions were treated with test chemical methyl methanesulfonate (CAS no. 66-27-3) (0.1, 0.2 and 0.3%) for 30 mins in the dark. Survival was evaluated by microscopic method, assessing the number of living cells (colonies) and dead cells due to the cytotoxic and mutagenic (lethal mutations) effects of test substance. The test was performed under static conditions for 30 mins with a 12 hr photoperiod. In the assay, the differences between the groups were analyzed using the standard t-test for paired observations. Statistical analyses were performed using ISI <R> Software, Version 2.0. Based on mortality of the test organism Chlamydomonas reinhardtii, the EC50 value was determined to be 1000 mg/l.

 

Another supporting study of short term toxicity Chlamydomonas reinhardtii study was carried out for 30 mins (Eva Miadokova et. al; 2005). The test organism used for the study was unicellular green alga C. reinhardtii recombination-repair-deficient strain uvs10. Algal cells were treated with test chemical methyl methanesulfonate (MMS) (0.1 - 0.5%) for 30 mins in the dark, and then plated on agar dishes. Survival was evaluated by microscopic method, which enabled to distinguish algal cells died due to cytotoxic and due to genotoxic (lethal mutations) effect of MMS. The test was performed under static conditions for 30 mins. For statistical analysis Wilcoxon’s two sample test was used. Based on mortality of the test organism Chlamydomonas reinhardtii, the EC50 value was determined to be 1000 mg/l. Thus based on the EC50 value, it can be concluded that the substance Methyl methanesulfonate can be considered as non-toxic to aquatic organisms and thus can be considered to be not classified as per the criteria of CLP regulation.

 

In addition to all the above supporting data, another short term toxicity to Chlamydomonas reinhardtii study was carried out for 30 mins (B. G. Hawks and R. W. Lee, 1976). The test organism used for the study green alga C. reinhardtii. Test chemical conc. used for the study was 1321.56 mg/l (12mM) or 2643.12 mg/l (24 mM), respectively. The test was performed under static conditions for 30 mins. Treatment with chemical Methyl methanesulfonate (MMS) (CAS no. 66-27-3) was performed on cells from different stages of synchronous growth after they were harvested and resuspended in 0.03 M, pH 7.0, phosphate buffer at a cell density of 107cells/ml. Mutagenesis was performed for 30 mins in darkness at 25°Cwith moderate shaking. Control samples not treated with MMS was prepared in a similar manner. Cell survival was determined during the period of 30 mins. Based on the percentage of cell survival of the test organism Chlamydomonas reinhardtii, the EC90 value for Methyl methanesulfonate was determined to be 1321.56 mg/l.

 

In a prediction done using the EPI Suite ECOSAR version 1.10 (EPI suite, 2017), the short term toxicity on green algae was predicted for test substance Methyl methanesulfonate (CAS no. 66 -27 -3). The 96 hours EC50 was estimated to be 2646.470 mg/L on algae for substance methyl methanesulphonate with growth inhibition effects.

 

Thus, based on the overall reported results for target chemical Methyl methanesulfonate (from peer reviewed journals), it can be concluded that the test substance Methyl methanesulfonate can be considered as non-toxic to aquatic environment and thus can be considered to be not classified as per the CLP classification criteria. 

 

Toxicity to microorganisms:

Based on the predicted data for the target chemical and experimental for read across chemicals study have been reviewed to determine the toxic nature of target chemical methyl methanesulfonate (MMS)(66-27-3) on the growth of microorganisms. The studies are as mentioned below: 

In the first key study for the target chemical methyl methanesulfonate (MMS) (66-27-3) from OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the six closest read across substances, toxicity on microorganism Tetrahymena pyriform is predicted for methyl methanesulfonate (66-27-3). Based on the growth rate inhibition, the IGC50 value was estimated to be 428.4 mg/l when methyl methanesulfonate (66-27-3) exposed to Tetrahymena pyriformis for 48hrs.

 

Similarly first study was supported by the second experimental study for read across chemical (106-65-0) from IUCLID dataset 2000, study was performed to determine the nature of chemical when comes in contact with test organisms. Aim of this study was to determine the toxic nature of chemical dimethyl succinate on the growth of microorganisms activated sludge, domestic. Study was conducted according to the OECD Guide–line 209 "Activated Sludge, Respiration Inhibition Test". After the exposure of chemical, effect concentration at which 50% inhibition was determine was observed. Thus after the exposure of chemical on domestic activated sludge, the EC20 was 29 mg/l, EC5080 was > 1000 mg/l and EC50 was determine to be > 1000 mg/l.

 

Based on the overall short term and long term studies on the growth and other activity of fishes, aquatic invertebrates and algae, it can be concluded that the chemical methyl methanesulfonate (MMS) (66-27-3) was toxic and can be consider to be classified in aquatic chronic category 2 as per the CLP classification criteria.