Esterification products of salicylic acid and C13-(branched) alcohols

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:

25 February 1997 to 10 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Direttiva CEE 92/69

Version / remarks:

Direttiva CEE 92/69

Principles of method if other than guideline:

In the Analysis and Biological Research Center Biolab Srl Italy, a study was performed in order to verify the mutagenic potential of test mateiral on strains of Salmonella typhimurium in accordance with "Direttiva CEE 92/69".
The solvents and doses used were chosen according to the product solubility. The study commenced on February 25, 1997 and ended on February 27, 1997.
In this report the concentrations are expressed in µg/0.1 ml (mcg of the test material/ 0.1 ml of DMSO solution placed on the plate).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tridecyl 2-hydroxybenzoate

EC Number:

947-975-7

Molecular formula:

C20H32O3

IUPAC Name:

tridecyl 2-hydroxybenzoate

Specific details on test material used for the study:

COSMACOL ESI 28L6
Composition (Cl2-Cl3) Alchyl ester of Salicilic acid
Receiving date January 27, 1997
Receiving n R00261.97

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

10,000 ug/l
1000 ug/l
100 ug/l
10 ug/l
0 (negative control)

Vehicle / solvent:

Dimeth7ylsulphoxide (DMSO)

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Characterization
Mutant strains of Salmonella typhimurium, TA1535, TA1537, TA1538, TA98 and TA100 were used

Maintenance of strains
The bacterial strains were preserved frozen. At the moment of use, the strains were subcultured 3 times consecutively on agar medium slants incubated at 37°C for 24 hours and therefore conserved at 4°C.

Genetic characteristics control
Each strain was checked before testing for the following genetic characteristics:

CHARACTERISTICS STRAIN

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

rfa = sensibility to crystalviolet
His" = histidine necessity
uvrB = sensibility to ultra-violet radiations
R.Amp = resistance to Ampicillin


CULTURE MEDIA AND REAGENTS

Minimal medium
Agar-Agar (Merck) 15.0 g
Glucose (Merck) 20.0 g
Magnesium sulfate (MgSo4 7H20 ) (Merck) 10.0 g
Citric acid monohydrate (Merck) 100.0 g
Potassium phosphate,dibasic (K.2HP0 4) (Merck) 500.0 g
(NaNH 4HP0 4.4H20 ) (Merck) 175.0 g
Distilled water q.b. (Merck) 1000 ml

Top agar
Agar-Agar (Merck) 6.0 g
Sodium chloride (Nacl) (Merck) 5.0 g
L-Histidine-HCl (0.5mM) (Merck) 100.0 g
D-Biotin (0.5mM) (Merck) 100.0 g

METABOLIC ACTIVATION SYSTEM

An enzimatic metabolic activation system (S9Mix) was prepared from the liver of male adult rats induced with "AROCLOR 1254".
AROCLOR was used as a dilution in soya bean oil containing 200 mg Aroclor/ml oil.
The S9Mix was checked for sterility.

APPARATUS AND GLASSWARE

Standard microbiological laboratory equipment and in particular the following was used:

Graduated pipettes Petri dishes
Test tubes Water bath
Vortex type agitator Laminar flow hoods

STUDY DESIGN
Test solution

The test material, was dissolved in Dimethylsulphoxide (DMSO) at a concentration of 100 mg/ml.

Dosage

The following concentrations expressed in µg/plate were used:

10.000
1000
100
10
0 (negative control)

Preliminary tests

Sterility test

Quantities of a solution of the test material were placed in plates containing minimal medium and in plates containing whole medium.
The occurrence of bacterial colonies was checked after 48 hours of incubation at 37°C.

Toxicity test

The test material was tested for toxicity on all the Salmonella typhimurium strains.
The toxic effects were determined by semiquantitative evaluation of the background lawn and of the number of spontaneous revertant colonies.

Positive controls

The positive controls were prepared at the following concentrations expressed in mg/plate:

Naazide 5
9-AA 40
2-NF 10
2-AA 1


TEST PROCEDURE

Plate test without metabolic activation

0 .1 ml of the appropriate dilution of the test solution and
0.1 ml of a bacterial suspension containing approximately 108 -109 cfu/ml 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.
The plates were incubated at 37°C for 48 - 72 hours. After incubation the revertant colonies obtained in the plates were counted.
Triplicate plates were used for the test material and two for the positive controls.

Plate test with metabolic activation

0.1 ml of the appropriate dilution 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.
The plates were incubated at 37°C for 48 - 72 hours. After incubation the revertant colonies obtained in the plates were counted.
Triplicate plates were used for the test material and two for the positive controls.

Evaluation criteria:

ACCEPTANCE CRITERIA

At the end of the assay, a sterility check on S9Mix must show less two viable colonies per 0.5 ml.

At the end of the test, a sterility check of the test material must show less than two viable colonies per plate.

The positive controls 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 ACCEPTABLE RANGE

TA 1535 20± 15
TA 1537 20± 15
TA 1538 15 ± 10
TA 98 40± 25
TA 100 150 ± 90

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST MATERIAL MUTAGEN/CITY (Table n° 1 and n° 2)

The test material at all concentration results NOT MUTAGENIC, both in the absence and in presence of the rat liver metabolizing system.

CONTROLS

Preliminary tests

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 in 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

All the positive controls induced a significant increase in the number of revertant colonies.

Sterility test

The sterility test performed on the test material and on S9Mix did not show any bacterial contamination

Applicant's summary and conclusion

Conclusions:

The test material at all concentration results NOT MUTAGENIC, both in the absence and in presence of the rat liver metabolizing system.

Executive summary:

In this test according to Directive CEE 92/69, the test material at all concentration is NOT MUTAGENIC, both in the absence and in presence of the rat liver metabolizing system.