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EC number: 940-877-5 | CAS number: -
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:
- 13 Mar - 17 Apr 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid
- EC Number:
- 940-877-5
- Cas Number:
- 2241455-89-8
- Molecular formula:
- C9H12N2O4
- IUPAC Name:
- 2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid
Constituent 1
Method
- Target gene:
- his operon
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Experiment 1 (standard plate test): 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Experiment 2 (pre-incubation test): 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without S9: sodium azide (TA 100, TA 1535); 4-nitro-o-phenylenediamine (TA 98; TA 1537); methylmethanesulfonate (TA 102); with S9: 2-aminoanthracene (all strains without TA102; TA 102)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: diminution of the background lawn or a reduction in the number of revertants
METABOLIC ACTIVATION SYSTEM:
The quality of the S9 homogenate was routinely tested in S. typhimurium using 2-aminoanthracene and benzo[a]pyrene - Evaluation criteria:
- A test item is considered mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
Biologically relevant means:
- number of reversion is at least twice the control (TA 98, TA 100, TA 102)
- number of reversion is at least thrice the control (TA 1535, TA 1537) - Statistics:
- Mean and standard deviation (no. of revertant colonies)
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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
- Key result
- Species / strain:
- S. typhimurium TA 102
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed in any tester strain
RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed in a range-finding assay (plate incorporation), tested up to 5000 µg/plate in TA 98 and TA 100.
COMPARISON WITH HISTORICAL CONTROL DATA:
The data were in accordance the historical control data.
Any other information on results incl. tables
Table 1: Test results of experiment 1 (standard plate test).
EXPERIMENT 1 (Standard Plate Test, SPT) |
|||||
S9-Mix |
Without
|
||||
Test item (µg/plate) |
TA 100 |
TA 1535 |
TA 98 |
TA 1537 |
TA 102 |
Water |
142±34.9 |
32±2.5 |
28±2.5 |
8±4.0 |
270±20.0 |
Control (DMSO) |
97±15.1 |
16±1.5 |
25±5.2 |
11±4.0 |
227±9.5 |
31.6 |
108±12.9 |
24±6.0 |
31±2.1 |
10±3.6 |
196±16.3 |
100 |
107±9.1 |
22±4.0 |
25±3.8 |
8±2.6 |
212±11.1 |
316 |
113±16.5 |
20±1.2 |
29±3.1 |
8±3.5 |
217±9.8 |
1000 |
103±3.2 |
19±3.8 |
21±8.7 |
10±3.2 |
234±19.5 |
2500 |
111±19.7 |
21±2.1 |
18±5.1 |
7±2.1 |
219±9.1 |
5000 |
103±11.0 |
16±1.7 |
24±6.1 |
10±3.2 |
225±4.9 |
NOPD (10 µg) |
- |
- |
424±34.8 |
86±10.5 |
- |
NaN3 (10 µg) |
1006±78.6 |
962±52.5 |
- |
- |
- |
MMS (1 µL) |
- |
- |
- |
- |
946±306.0 |
|
|
|
|
|
|
S9-Mix |
With |
||||
|
|
|
|
|
|
Test item (µg/plate) |
TA 100 |
TA 1535 |
TA 98 |
TA 1537 |
TA 102 |
Water |
129±12.5 |
25±1.5 |
34±2.1 |
13±2.6 |
309±31.2 |
NC (DMSO) |
123±3.6 |
26±2.3 |
22±9.6 |
8±1.7 |
259±11.0 |
31.6 |
123±5.5 |
24±6.4 |
30±4.6 |
8±1.7 |
263±39.7 |
100 |
120±7.9 |
26±1.7 |
27±2.9 |
10±5.5 |
277±13.2 |
316 |
117±18.9 |
20±4.6 |
31±4.7 |
11±1.7 |
249±20.5 |
1000 |
119±9.1 |
23±2.6 |
27±4.6 |
10±2.3 |
241±21.8 |
2500 |
118±19.6 |
28±2.6 |
28±3.6 |
11±2.1 |
279±10.1 |
5000 |
108±0.7 |
20±4.0 |
33±10.6 |
8±3.8 |
275±13.3 |
2-AA (2.5 µg) |
1808±119.8 |
166±17.5 |
3160±201.7 |
375±11.0 |
547±68.7 |
Table 2: Test results of experiment 2 (preincubation assay).
EXPERIMENT 2 (preincubation assay) |
|||||
S9-Mix |
Without
|
||||
Test item (µg/plate) |
TA 100 |
TA 1535 |
TA 98 |
TA 1537 |
TA 102 |
Water |
135±7.6 |
16±2.5 |
26±9.0 |
6±2.9 |
287±14.9 |
Control (DMSO) |
126±9.5 |
12±3.2 |
18±1.0 |
5±2.3 |
221±2.5 |
31.6 |
92±6.7 |
17±3.1 |
21±3.5 |
8±3.5 |
238±14.2 |
100 |
116±25.4 |
13±3.2 |
19±6.1 |
9±3.5 |
254±4.9 |
316 |
98±12.1 |
12±4.7 |
21±1.5 |
5±1.5 |
229±12.2 |
1000 |
100±12.5 |
12±1.5 |
23±6.0 |
6±1.2 |
248±13.7 |
2500 |
101±5.1 |
14±5.1 |
22±4.0 |
5±5.1 |
258±33.2 |
5000 |
100±14.2 |
16±2.9 |
22±2.9 |
5±2.9 |
255±28.0 |
NOPD (10 µg) |
- |
- |
470±35.9 |
61±30.3 |
- |
NaN3 (10 µg) |
1065±64.5 |
1152±105.1 |
- |
- |
- |
MMS (1 µL) |
- |
- |
- |
- |
1851±10.4 |
S9-Mix |
With |
||||
|
|
|
|
|
|
Test item (µg/plate) |
TA 100 |
TA 1535 |
TA 98 |
TA 1537 |
TA 102 |
Water |
166±6.7 |
11±4.6 |
23±1.5 |
11±1.7 |
412±11.6 |
NC (DMSO) |
130±0.0 |
10±1.2 |
32±2.0 |
5±3.1 |
323±6.1 |
31.6 |
146±13.7 |
14±4.6 |
33±4.5 |
5±2.0 |
296±15.0 |
100 |
146±7.8 |
11±3.6 |
28±7.8 |
5±1.0 |
303±44.8 |
316 |
121±20.6 |
17±4.6 |
25±5.7 |
4±0.6 |
310±12.1 |
1000 |
132±4.7 |
12±3.5 |
25±2.5 |
7±0.0 |
298±17.7 |
2500 |
149±9.5 |
10±2.3 |
34±2.5 |
6±2.1 |
309±26.6 |
5000 |
139±3.5 |
10±4.9 |
20±2.9 |
6±2.1 |
319±42.2 |
2-AA (2.5 µg) |
2208±69.0 |
194±17.5 |
1949±348.5 |
186±30.4 |
671±23.7 |
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results:
negative
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