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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:
1993
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

Constituent 1
Chemical structure
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-methylpropionate
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:

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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