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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-06-22-1992-06-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No testing of phenotypic characteristics mentioned (no ampicillin resistance, sensitivity to crystal violet and ultraviolet light), no justification for missing follow-up experiment given.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Butanedioic acid, 2,3-dihydroxy- [R-(R*,R*)]-,C12-13-branched alkyl esters
EC Number:
947-004-7
Molecular formula:
C28H56O6 - C29H58O6 - C30H60O6
IUPAC Name:
Butanedioic acid, 2,3-dihydroxy- [R-(R*,R*)]-,C12-13-branched alkyl esters
Test material form:
liquid
Details on test material:
product

Method

Target gene:
TA 1535 rfa His- uvrB
TA 1537 rfa His- uvrB
TA 1538 rfa His- uvrB
TA 98 rfa His- uvrB R. Amp
TA 100 rfa His- uvrB R.Amp
Species / strain
Species / strain / cell type:
S. typhimurium, other: Strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, derived from the livers of male adult rats induced with Aroclor 1254. AROCLOS was used as a dilution in soya bean oil containing 200 mg Aroclor/ml. The S9 mix was checked for sterility.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 1, 10, 100, 1000 and 10000 µg/plate
Concentration range in the main test (without metabolic activation): 1, 10, 100, 1000 and 10000 µg/plate
Vehicle / solvent:
- Solvent: DMSO
- Justification for choice of solvent/vehicle: no data
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene 1 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

Test solution: The test material was dissolved in DMSO at a concentration of 100 mg/ml.

Plate test without metabolic activation:
0.1 ml of the test solution and 0.1 ml of a bacterial suspension containing approximately 10exp8 - 10exp9 cfu/ml were added in this sequence to 2 ml of molten top agar. The ingredients were thouroughly mixed and immediately poured into plates containing minimal medium. At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.

Plate test with metabolic activation
0.1 ml of the test solution, 0.1 ml of bacterial suspensions and 0.5 ml of the metabolic activation system were added in this sequence to 2 ml of molten top agar. The ingredients were thoroughly mixed and immediately poured into plates containing minimal medium. At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 - 72 h
After incubation the revertant colonies obtained in the plates were counted.

NUMBER OF REPLICATIONS: triplicates plates were used for the test material, two for positive controls




DETERMINATION OF CYTOTOXICITY
- Method: determination of toxicity by semiquantitative evaluation of the background lawn and the number of spontaneous revertant colonies

OTHER EXAMINATIONS:
Sterility test
Evaluation criteria:
- at the end of the assay, a sterility check on S9 mix must show less than two viable colonies per 0.5 mL
- at the end of the test, a sterility check of the test material nust show less than two viable colonies per plate
- the positive conrols must produce at least a threefold increase in the number of revertant colonies with regard to the mean value for the respective negative control
- the mean number of spontaneous revertant colonies in the negative controls must correspond to the following values:
strain TA 1535: 20 ± 15; strain TA 1537: 20 ± 15; strain TA 1538: 15 ± 10; strain TA 98: 40 ± 25; strain TA 100: 150 ± 90
Statistics:
no statistics performed

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: Salmonella typhimurium: strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: Salmonella typhimurium: strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 10000 µg/plate
Vehicle controls validity:
other: vehicle controls valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- Preliminary test: The genetic characteristics of the bacterial strains were found to be unaltered.
- Test material toxicity: The test material did not induce toxic effects at a concentration of 10 mg/plate both in the absence and presence of the metabolic activation system.
- Negative control: The number of revertant colonies on the negative control plates fell within the normal range.
- Positive control: The positive controls induced an increase in the number of colonies.
- Sterility test: The sterility test performed on the test material and on S9 mix did not show any bacterial contamination.

Any other information on results incl. tables

Table #1: Salmonella typhimurium reverse mutation assay without metabolic activation
Concentration
[µg/plate]
Revertant colonies / plate
mean ± SD
Strain TA 1535 Strain TA 1537 Strain TA 1538 Strain TA 98 Strain TA 100
0 12 ± 3 17 ± 2 13 ± 3 9 ± 4 105 ± 19
1 14 ± 3 16 ± 5 13 ± 2 12 ± 2 166 ± 23
10 16 ± 7 17 ± 2 12 ± 2 10 ± 2 101 ± 21
100 18 ± 6 16 ± 2 9 ± 1 16 ± 5 114 ± 17
1000 22 ± 8 17 ± 1 13 ± 4 15 ± 2 112 ± 16
10000 15 ± 3 16 10 ± 4 13 ± 2 114 ± 14
Positive control 433 ± 47 96 ± 283 264 ± 31 385 ± 18 152 ± 37
Table #2: Salmonella typhimurium reverse mutation assay with metabolic activation
Concentration
[µg/plate]
Revertant colonies / plate
mean ± SD
Strain TA 1535 Strain TA 1537 Strain TA 1538 Strain TA 98 Strain TA 100
0 25 ± 3 24 ± 2 19 ± 2 16 ± 3 113 ± 12
1 22 ± 3 21 ± 2 19 ± 2 19 ± 6 121 ± 13
10 25 ± 4 19 ± 3 15 ± 2 19 ± 4 121 ± 19
100 19 ± 2 24 ± 5 18 ± 1 18 ± 4 117 ± 17
1000 21 ± 1 21 ± 4 15 ± 5 17 ± 2

121 ± 6

10000

26 ± 3

 22 ± 3

16 ± 4

17 ± 4

133 ± 17

Positive control

66 ± 3

480 ± 23

658 ± 14

216 ± 5

335 ± 21

SD = Standard Deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test material induced the development of as many revertant colonies as those induced by the negative control, both in the absence and in presence of the rat liver metabolizing system, at the maximum dose tested (10 mg/plate).

The test material did not induce an increase in the number of revertant colonies compared to the negative controls and was considered as non-mutagenic in this test-system.
Executive summary:

A test was performed to verify the mutagenic potential of the test material Butanedioic acid, 2,3-dihydroxy-[R-(R*, R*)], C12-13-branched alkyl esters on Salmonella typhimurium.

The test was carried out on 5 strains of Salmonella typhimurium (TA 1535, TA 1537, TA1538, TA 98, TA 100). The mutagenic activity of the material was assessed by comparing the number of revertant colonies induced with the positive control. This activity was tested in the presence and absence of a metabolizing system and done directly in a petri dish. The test material was tested at concentrations of 100000, 10000, 1000, 100 and 10 µg/mL corresponding to doses of 10000, 1000, 100, 10 and 1 µg/petri dish. The results of the study can be summatized as follows: The test material at maximum concentration 10 mg/petri dish produced an increase in number of revertant colonies similar to the negative control so the test material Butanedioic acid, 2,3-dihydroxy-[R-(R*, R*)], C12-13-branched alkyl esters is unable to induce mutation under these conditions.

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