Methyl methanesulphonate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: refer below principle

Principles of method if other than guideline:

Combine multigeneration reproductive and genotoxicity study of methyl methanesulfonate in zebrafish

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Methyl methanesulfonate
- Molecular formula (if other than submission substance): C2H6O3S
- Molecular weight (if other than submission substance): 110.1324 g/mol
- Smiles notation (if other than submission substance): COS(=O)(=O)C
- InChI: 1S/C2H6O3S/c1-5-6(2,3)4/h1-2H3
- Substance type: Organic
- Physical state: Liquid

Analytical monitoring:

yes

Details on sampling:

Details on sampling
- Concentrations: 0, 0.2 and 0.4 mg/L
- Sampling method: 10 mL tap
water containing 0, 0.2 and 0.4 mg/L MMS in glass beakers at 26 ± 1 ˚C
- Sample storage conditions before analysis: 26 ± 1 ˚C and a photoperiod of 12 h:12 h

Vehicle:

yes

Details on test solutions:

Details on test solutions
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mL tap water containing 0, 0.2 and 0.4mg/L MMS in glass beakers at 26 ± 1˚C and a photoperiod of 12 h: 12 h, which was maintained throughout the whole experiment.
- Eluate: No data available
- Differential loading: Yes, flow of stock solutions (52, 104, 208 mg/L MMS) was adjusted by means of peristaltic pumps
- Controls: Tap water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Water
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 10 L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No data available

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio, West Aquarium wild
- Source: Laboratory
- Age at study initiation (mean and range, SD): 220 days post fertilization
- Length at study initiation (length definition, mean, range and SD): No data available
- Weight at study initiation (mean and range, SD): No data available
- Method of breeding: Fertilization
- Feeding during test: Yes
- Food type: Artemia nauplii, powder food and flakes.
- Amount: No data available
- Frequency: Artemia nauplii fed once, powder food, three times and flakes two times a day


ACCLIMATION
- Acclimation period: 1–2 h
- Acclimation conditions (same as test or not): Same
- Type and amount of food: Artemia nauplii fed once, powder food, three times and flakes two times a day
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): No data available

QUARANTINE (wild caught)
- Duration: No data available
- Health/mortality: No data available

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

30 d

Post exposure observation period:

370 days post fertilization

Hardness:

12–18˚dH

Test temperature:

25 ± 2 ˚C

pH:

7.0–8.3

Dissolved oxygen:

No data available

Salinity:

No data available

Nominal and measured concentrations:

0.2 and 0.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Tank
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 10 L
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): Renewed every 48 hours
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): Triplicate
- No. of vessels per control (replicates): Triplicate
- No. of vessels per vehicle control (replicates): Triplicate
- Biomass loading rate: No data available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: No data available
- Total organic carbon: No data available
- Particulate matter: No data available
- Metals: No data available
- Pesticides: No data available
- Chlorine: No data available
- Alkalinity: No data available
- Ca/mg ratio: No data available
- Conductivity: 522–828 lS/cm
- Culture medium different from test medium: No data available
- Intervals of water quality measurement: Weekly

OTHER TEST CONDITIONS
- Adjustment of pH: No data available
- Photoperiod: 12 h:12 h
- Light intensity: No data available

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, Growth, Body weight, length, Fertility and fertilization, Teratogenicity in embryos and early larval stages, Genotoxicity and histopathology were measured.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: No data available
- Justification for using less concentrations than requested by guideline: Since all MMS-exposed spawning groups failed to spawn, and fish exposed to 4 mg/L MMS also showed mortalities, MMS concentrations were reduced from 2 and 4 to 0.2 and 0.4 mg/L, respectively
- Range finding study: No data available
- Test concentrations: 0.2 and 0.4 mg/L
- Results used to determine the conditions for the definitive study: No data available

Reference substance (positive control):

not specified

Key result

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Effect on survival , growth, length of fish, Fertility and fertilization, Teratogenicity in embryos and early larval stages, Genotoxicity and Histopathology

Remarks on result:

other: At 300 dpf, all remaining individuals exposed to 0.4 mg/L MMS were sacrificed because of mortality, macroscopic pathology and consistent lack of spawn. F0 control fish as well as fish exposed to 0.2 mg/L MMS were sacrificed at 370 dpf.

Details on results:

- Mortality/survival at embryo and larval stages: Yes, 50 % mortality was observed.
- Overall mortality/survival: 53 % were observed as compared to control.
- Days to hatch and numbers hatched: 96 days
- Data for length and weight of surviving fish:
Body weight: Significant increase in body weight was observed in 0.2 mg/L treated fish as compared to control.

Length: No effect on total length of F1 fish were observed in 0.2 mg/L dose as compared to control

- Type of and number with morphological abnormalities: Earlier spawn were observed in 0.2 mg/L treated fish as compared to control.

- Type of and number with behavioural abnormalities: No data available

- Other biological observations:
Fertilization: In 0.2 mg/L dose group, increase egg productions relative to the controls were observed.

Teratogenicity in embryos and early larval stages: Significantly more sublethal teratogenic effects in embryos and larvae were observed in 0.2 mg/L treated fish as compared to control.

Genotoxicity:
Percentage of DNA significantly elevated in tail and liver cells from sexes as well as female gills and male gonads of 0.2 mg/L treated fish as compared to control.

Histopathology:
Micronucleus frequencies were significantly increased in liver, gills and gonads of both male and female fish in 0.2 mg/L treated fish as compared to control.

- Effect concentrations exceeding solubility of substance in test medium: No data available

- Incidents in the course of the test which might have influenced the results: No data available

Results with reference substance (positive control):

No data available

Reported statistics and error estimates:

No data available

For F0 generation 0, 2, 4 and 8 mg/L dose were used and for F1 generation 0, 0.2 and 0.4 mg/L dose were used.

Validity criteria fulfilled:

yes

Conclusions:

In a long term Combine multigeneration reproductive and genotoxicity study, Danio rerio, treated with Methyl methanesulfonate (MMS) in the concentration of 0, 0.2 and 0.4 mg/L for 30 days. In the study EC50 was considered to be 0.2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS).

Executive summary:

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. 

Description of key information

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. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.2 mg/L

Additional information

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.