Bis(C12-C13)alkyl-2-hydroxybutandioate

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:

June 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study but some of the testing laboratory's own acceptance criteria not met, no justification for missing follow-up experiment

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

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Cosmacol EMI
- Substance type: pure active scubstance
- Physical state: waxy solid

Method

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

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:

other: without S9 mix: 9-Aminoacridine (40 µg/plate) with strain TA1537, Sodium azide (5 µg/plate) with strains TA 1535 and TA 100, 2-Nitrofluorene (10 µg/plate) with strains TA 1538 and TA 98; with S9 mix: 2-Aminoanthracene (1 µg/plate) with all tester strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates plates for the test material and negative control, 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

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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:

no cytotoxicity

Vehicle controls validity:

other: vehicle controls valid with the exception of strain TA 98

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 with the exception of strain TA 98 without metabolic activation. This negative control (9 ± 2) was below the cited normal range of 40 ± 25 revertant colonies.
- Positive control: The positive controls induced a at least threefold increase in the number of colonies with the exception of the positive controls with strain TA 98 both with and without S9 mix. In these cases the increase of revertant colonies was not threefold (without S9 mix: 2.4-fold, with S9 mix: 2.6-fold).
- 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	23 ± 6	11 ± 3	9 ± 3	9 ± 2	64 ± 10
1	23 ± 2	23 ± 4	12 ± 3	13 ± 1	93 ± 10
10	24 ± 8	14 ± 5	11 ± 3	11 ± 2	75 ± 20
100	20 ± 5	18 ± 3	10 ± 2	12 ± 3	97 ± 16
1000	19 ± 6	13 ± 3	10 ± 5	9 ± 2	86 ± 10
10000	19 ± 3	18 ± 3	9 ± 3	11 ± 1	92 ± 15
Positive control	433 ± 47	96 ± 3	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	22 ± 4	11 ± 2	21 ± 5	26 ± 8	129 ± 13
1	27 ± 1	14 ± 6	18 ± 7	25 ± 5	137 ± 20
10	20 ± 5	20 ± 3	19 ± 4	23 ± 8	149 ± 17
100	25 ± 6	18 ± 4	13 ± 5	28 ± 2	133 ± 20
1000	26 ± 9	15 ± 4	11 ± 1	24 ± 7	193 ± 13
10000	17 ± 5	24 ± 5	14 ± 5	22 ± 5	166 ± 16
Positive control	66 ± 3	480 ± 23	206 ± 14	216 ± 5	335 ± 21

SD = Standard Deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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 potentiol of the test material Bis(C12 -C13)alkyl-2 -hydroxybutandioate on Salmonella typhimurium according to Ames B.N., McCann J. and Yamasaki E. (1975).

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: Teh 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 Bis(C12-C13)alkyl-2-hydroxybutandioate is unable to induce mutation under these conditions.