tert-pentyl 2-ethylperoxyhexanoate

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:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: GLP, OECD 471 Guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-pentyl 2-ethylperoxyhexanoate

EC Number:

211-687-3

EC Name:

tert-pentyl 2-ethylperoxyhexanoate

Cas Number:

686-31-7

Molecular formula:

C13H26O3

IUPAC Name:

2-methylbutan-2-yl 2-ethylhexaneperoxoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

The test substance was identified as follows:
. name: t-AMYL PEROXYETHYL-2-HEXANOATE
. batch number: 616-9809-715 (19/09/98)
. description:
- at receipt: whitish liquid
-on the test article description: colourless or slightly yellowish liquid
. container: one plastic flask
. date of receipt: 16 December 1998
. storage conditions: at +4°C and protected from light
. expiry date: June 1999.

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Metabolic activation system:

: Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with Arochlor 1254

Test concentrations with justification for top dose:

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.
Without S 9 the following doses were chosen: 156.25, 312.5, 625, 1250 and 2500 pg/plate: for all tester strains in both experiments.
With S9: The selected treatment-levels were as follows: 156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 1535, TA 100 and TA 102 strains in the first experiment / 78.125, 156.25, 312.5, 625 and 1250 µg/plate: for the TA 1537 and TA 98 strains in the first
experiment / 312.5, 625, 1250, 2500 and 3125 µg/plate: for the TA 1535, TA 100 and TA 102 strains in the second experiment / 312.5, 625, 750, 1250 and 1500 µg/plate: for the TA 1537 and TA 98 strains in the second experiment.

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below, details on test system and conditions

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY (preliminary range-finding test)
- Test: in TA 98, TA 100 and TA 102 strains, with or without S9 mix ; 6 dose-levels (one plate/dose level)
- Method: relative total growth (decrease in the number of revertant colonies and/or a thinning of the bacterial lawn);

EXPERIMENTS
Number of independent experiments: 2.

METHOD OF APPLICATION:
* Direct plate incorporation method: for preliminary test and first experiment
* Preincubation: for the second experiment

DURATION
- Preincubation period: 60 min
- Exposure duration: 48-72H

NUMBER OF REPLICATIONS: triplicates

CONTROLS
* Without S9 mix
- Sodium azide (NAN3): 1 µg/plate for TA 1535; TA 100 strains
- 9-Aminoacridine (9AA): 50 µg/plate for TA 1537 strain
- 2-Nitrofluorene (2NF): 0.5 µg/plate for TA 98 strain
- Mitomycin C (MMC): 0.5 µg/plate for TA 102 strain
* With S9 mix:
- 2-Anthramine (2AM): 2 µg/plate for TA 1535; TA 1537; TA 98; TA 100 strain and 2AM: 10 µg/plate for TA 102 strain

Evaluation criteria:

Reproducible increase in the number of revertant colonies (2-fold for TA98/TA100 and TA102, 3-fold for TA 1535/TA 1537) compared with vehicle controls in any strain at any dose-level and/or evidence of a dose-relationship.
Reference to historical data and consideration to biological relevance may also be taken into account.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY:
- Results on solubility: The test substance was freely soluble in the vehicle at 50 mg/ml. Consequently, with a maximum dose volume of 100 µl/plate, the dose-levels for the preliminary toxicity test were: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
- Results on cytotoxicity: A slight to strong emulsion was observed in the Petri plates when scoring the revertants at dose­
levels 2500 µg/plate. Without S9 mix, a slight toxicity was noted towards the TA 98 and TA 102 strains at dose-Ievels of 500 µg/plate and 1000 pg/plate, respectively. In the TA 100 strain, a slight to moderate toxicity was induced at dose-levels 100 µg/plate. With S9 mix, the test substance was totally toxic at dose-levels of 5000 µg/plate (TA 100 and TA 102 strains) or 2500 pg/plate (TA 98 strain). In addition in the TA 98 strain, a slight thinning of the bacterial lawn was noted at 1000 µg/plate. In the TA 102 strain, with S9 mix, a 2.6-4.7 fold increase in the number of revertants was noted at dose-levels of between 500 and 2500 µg/plate.




Mutagenicity results are presented in tables 1 to 4.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results: see attached doc

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Tert-amyl peroxy-2-ethylhexanoate shown mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The potential of the test item Tert-amyl peroxy-2-ethylhexanoateto induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102) was evaluated in accordance with the international guidelines (OECD 471, Commission Directive No. B13/14) in compliance with the Principles of Good Laboratory Practice.

The test item was tested in two independent experiments, with and without a metabolic activation system.

Both experiments were performed according to the direct plate incorporation method except the second with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Bacterias were exposed to the test item at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix in TA98, 100, 1537 and 1538. The test item induced a mutagenic activity in TA 102 but only with S9.

Tert-amyl peroxy-2-ethylhexanoate shown mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in TA 102 with S9.