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EC number: 218-301-2 | CAS number: 2110-78-3
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1993-07-05 - 1993-07-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 993
- Report date:
- 1993
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- method described in Article 34-3 of the Ordinance in Industrial Safety and Health
- Deviations:
- not specified
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Methyl 2-hydroxy-2-methylpropionate
- EC Number:
- 218-301-2
- EC Name:
- Methyl 2-hydroxy-2-methylpropionate
- Cas Number:
- 2110-78-3
- Molecular formula:
- C5H10O3
- IUPAC Name:
- methyl 2-hydroxy-2-methylpropanoate
- Test material form:
- liquid
- Details on test material:
- - State of aggregation: Colorless transparent liquid
- Density: 1.023 g/cm³ (25°C)
- Flash point: 137°C
- impurity: Water (0.1 wt%)
- Molecular weight: 118.12
- Soluble in water and oil
- Other:
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Institute for fermentation
- Storage temperature: -80°C
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMSO
- Properly maintained: yes
- Storage temperature: -80°C
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Institute for fermentation
- Storage temperature: -80°C
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMSO
- Properly maintained: yes
- Storage temperature: -80°C
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- dose finding study: 10, 50, 100, 500, 1000, and 5000 µg/plate
main study: 156, 313, 625, 1250, 2500, and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: sterile ion-exchange distilled water
Controlsopen allclose all
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- N-ethyl-N-nitro-N-nitrosoguanidine
- furylfuramide
- other: 2-Aminoanthracene (2-AA) / and ICR-191
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
Cell suspension: 0.1 mL
Dosing solution: 0.1 mL
Na-phosphate buffer solution: 0.5 mL
Soft agar: 2 mL
Temperatuer: 37°C
DURATION
- Preincubation period: 11 hrs in Nutrient Broth No. 2 (OXOID)
- Pre-incubation study: 20 min (37°C)
- Exposure duration: 48 hrs - Evaluation criteria:
- Number of revertant colonies.
- Statistics:
- Mean revertant colony count were determined for each dose point.
Results and discussion
Test results
- Key result
- Species / strain:
- other: Salmonella typhimurium TA100, TA98, TA1535 and TA1537, and Escherichia coli WP2uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Table 3: Study results (dose-finding study), Name of test substance: methyl 2 -hydroxyisobutyrate
| Presence or absence of metabolic activation |
Concentration of test substance (µg/plate) |
Revertant colony count (per plate) | |||||||||||||||
| Base-pair substitution | Frame shift | ||||||||||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||||||||
| S9 Mix: (-) | Solvent control | 122 | 122 | (122) | 6 | 10 | (8) | 23 | 28 | (26) | 22 | 28 | (25) | 18 | 8 | (13) | |
| 10 | 112 | 108 | (110) | 9 | 9 | (9) | 19 | 26 | (23) | 24 | 21 | (23) | 12 | 5 | (9) | ||
| 50 | 123 | 99 | (111) | 3 | 7 | (5) | 38 | 40 | (39) | 20 | 29 | (25) | 8 | 10 | (9) | ||
| 100 | 102 | 108 | (105) | 2 | 4 | (3) | 32 | 25 | (29) | 20 | 18 | (19) | 15 | 8 | (12) | ||
| 500 | 130 | 114 | (122) | 5 | 11 | (8) | 31 | 33 | (32) | 23 | 27 | (25) | 8 | 11 | (10) | ||
| 1000 | 117 | 103 | (110) | 6 | 6 | (6) | 29 | 28 | (29) | 24 | 30 | (27) | 7 | 7 | (7) | ||
| 5000 | 105 | 97 | (101) | 10 | 7 | (9) | 28 | 36 | (32) | 25 | 27 | (26) | 9 | 11 | (10) | ||
| S9 Mix: (+) | Solvent control | 121 | 125 | (123) | 7 | 7 | (7) | 27 | 15 | (21) | 30 | 36 | (33) | 10 | 16 | (13) | |
| 10 | 122 | 129 | (126) | 10 | 7 | (9) | 22 | 16 | (19) | 31 | 25 | (28) | 19 | 15 | (17) | ||
| 50 | 110 | 116 | (113) | 10 | 4 | (7) | 18 | 24 | (21) | 32 | 31 | (32) | 12 | 14 | (13) | ||
| 100 | 100 | 100 | (100) | 9 | 3 | (6) | 19 | 25 | (22) | 23 | 39 | (31) | 18 | 20 | (19) | ||
| 500 | 127 | 109 | (118) | 10 | 4 | (7) | 32 | 24 | (28) | 19 | 33 | (26) | 13 | 15 | (14) | ||
| 1000 | 102 | 122 | (112) | 7 | 12 | (10) | 22 | 25 | (24) | 33 | 35 | (34) | 13 | 1 | (12) | ||
| 5000 | 109 | 109 | (109) | 10 | 9 | (10) | 31 | 26 | (29) | 39 | 28 | (34) | 9 | 20 | (15) | ||
| Positive control |
S9 Mix not required | Name | AF-2 | NaN3 | ENNG | 2-NF | ICR-191 | ||||||||||
| Concentration (µg/plate) | 0.01 | 0.5 | 2 | 1 | 1 | ||||||||||||
| Colony count/plate |
465 | 463 | (464) | 213 | 277 | (245) | 319 | 344 | (332) | 628 | 613 | (621) | 2070 | 2596 | (2333) | ||
| S9 Mix required | Name | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA | |||||||||||
| Concentration (µg/plate) | 1 | 2 | 10 | 0.5 | 2 | ||||||||||||
| Colony count/plate |
904 | 960 | (932) | 200 | 221 | (211) | 876 | 780 | (828) | 469 | 331 | (400) | 134 | 154 | (144) | ||
NB: Figures in parentheses indicate the mean colony count on each plate
Table 4: Study results (main study), Name of test substance: methyl 2-hydroxyisobutyrate
| Presence or absence of metabolic activation |
Concentration of test substance (µg/plate) |
Revertant colony count (per plate) | |||||||||||||||
| Base-pair substitution | Frame shift | ||||||||||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||||||||
| S9 Mix: (-) | Solvent control | 119 | 136 | (128) | 6 | 10 | (8) | 21 | 22 | (22) | 22 | 13 | (18) | 7 | 3 | (5) | |
| 156 | 122 | 132 | (127) | 10 | 5 | (8) | 18 | 22 | (20) | 11 | 15 | (13) | 4 | 3 | (4) | ||
| 313 | 95 | 114 | (105) | 9 | 15 | (12) | 22 | 29 | (26) | 13 | 13 | (13) | 8 | 7 | (8) | ||
| 625 | 110 | 92 | (101) | 8 | 11 | (10) | 26 | 20 | (23) | 18 | 20 | (19) | 7 | 7 | (7) | ||
| 1250 | 121 | 116 | (119) | 7 | 6 | (7) | 28 | 22 | (25) | 19 | 18 | (19) | 3 | 6 | (5) | ||
| 2500 | 105 | 110 | (108) | 8 | 6 | (7) | 28 | 16 | (22) | 16 | 18 | (17) | 4 | 4 | (4) | ||
| 5000 | 107 | 116 | (112) | 12 | 14 | (13) | 21 | 19 | (20) | 14 | 19 | (17) | 6 | 7 | (7) | ||
| S9 Mix: (+) | Solvent control | 108 | 116 | (112) | 7 | 7 | (7) | 27 | 9 | (18) | 22 | 29 | (26) | 19 | 13 | (16) | |
| 156 | 102 | 118 | (110) | 5 | 12 | (9) | 21 | 23 | (22) | 26 | 19 | (23) | 16 | 18 | (17) | ||
| 313 | 115 | 123 | (119) | 10 | 9 | (10) | 13 | 18 | (16) | 25 | 24 | (25) | 11 | 13 | (12) | ||
| 625 | 109 | 99 | (104) | 10 | 8 | (9) | 19 | 13 | (16) | 23 | 23 | (23) | 23 | 14 | (19) | ||
| 1250 | 100 | 98 | (99) | 11 | 12 | (12) | 27 | 16 | (22) | 24 | 27 | (26) | 10 | 12 | (11) | ||
| 2500 | 112 | 100 | (106) | 8 | 7 | (8) | 27 | 27 | (27) | 27 | 28 | (28) | 13 | 24 | (19) | ||
| 5000 | 109 | 99 | (104) | 9 | 9 | (9) | 25 | 18 | (22) | 24 | 33 | (29) | 10 | 14 | (12) | ||
| Positive control |
S9 Mix not required | Name | AF-2 | NaN3 | ENNG | 2-NF | ICR-191 | ||||||||||
| Concentration (µg/plate) | 0.01 | 0.5 | 2 | 1 | 1 | ||||||||||||
| Colony count/plate |
515 | 521 | (518) | 213 | 277 | (245) | 412 | 474 | -443 | 634 | 593 | (614) | 1706 | 1468 | (1587) | ||
| S9 Mix required | Name | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA | |||||||||||
| Concentration (µg/plate) | 1 | 2 | 10 | 0.5 | 2 | ||||||||||||
| Colony count/plate |
788 | 822 | (805) | 200 | 221 | (211) | 826 | 784 | (805) | 425 | 326 | (376) | 205 | 176 | (191) | ||
NB: Figures in parentheses indicate the mean colony count on each plate
Applicant's summary and conclusion
- Conclusions:
- Results obtained in the present mutagenicity study indicated that the test substance methyl 2-hydroxyisobutyrate shows no mutagenic potential since the revertant colony counts determined for each tester strain in both the presence (metabolic activation method) and absence (non-metabolic activation method) of S9 mix were within the range of spontaneous revertant colony counts.
- Executive summary:
The test substance methyl 2-hydroxyisobutyrate was evaluated in an in vitro bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2uvrA as tester strains in accordance with the Ames test method described in Article 34 -3 of the Ordinance on Industrial Safety and Health (comparable to OECD Guideline 471)
with and without metabolic activation (S9 mix) at concentrations in the main study of 156, 313, 625, 1250, 2500, and 5000 µg/plate. Results obtained in the present mutagenicity study indicated that the test substance shows no mutagenic potential since the revertant colony counts determined in both the presence (metabolic activation method) and absence (non-metabolic activation method) of S9 mix were within the range of spontaneous revertant colony counts.
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