Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

EC Number:

940-594-7

Molecular formula:

C52H86O4S3 , C37H58O3S2

IUPAC Name:

Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

Test material form:

liquid

Details on test material:

Intended use: Antioxidant for plastics
Appearance: Brown liquid
Storage conditions: Room temperature protected from light
Supplier: Sponsor
Lot number: 102Y1
Expiry date: 30 April 2014
Purity 98.8%

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:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

The highest concentration of AO-26 tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with acetone to produce a series of lower concentrations, separated by approximately half-log10 intervals.

5 μg
15 μg
50 μg
150 μg
500 μg
1500 μg
5000 μg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone;
- Justification for choice of solvent/vehicle: The Sponsor indicated that AO-26 was poorly soluble in water and in dimethyl sulphoxide but was soluble in acetone. Acetone (analytical reagent grade) was, therefore, used as the vehicle for this study.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix

Controls 3

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix

Controls 4

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

In the absence of S9 mix

Controls 5

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9 mix

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

First test
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was acetone. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum
concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least one precipitating concentration should be included in the second test, unless otherwise justified by the Study Director.

Rationale for test conditions:

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for acetone, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

Statistics:

No statistical analysis is performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Applicant's summary and conclusion

Conclusions:

It was concluded that AO-26 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of AO-26, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to AO-26 diluted in acetone. Acetone was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Both tests were standard plate incorporation assays.

Concentrations of AO-26 up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

No signs of toxicity towards the tester strains were observed in either mutation test following exposure to AO-26.

No evidence of mutagenic activity was seen at any concentration of AO-26 in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

It was concluded that AO-26 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.