Butanedioic acid, 2,3-dihydroxy- [R-(R*,R*)]-,C12-13-branched alkyl esters

Genetic toxicity: in vitro

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

Test material information

Constituent 1

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 results

Test results 1

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

Test results 2

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.