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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro tests:

Ames test:

The potential of the test item Tert-amyl peroxy-2-ethylhexanoatet to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102) was evaluated in accordance with OECD 471 guideline and was GLP compliant (Haadouk, 1999). 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 strains TA 1535, 1537, 100 and 98. Mutagenic effect was observed in TA 102 but only with S9.

Mouse Lymphoma Assay:

Tert-pentyl-2 -ethylhexanoate, was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The study was designed to comply with the OECD 476 guideline (adopted July 1997) and was in compliance with GLP (, 2012). A main assay for mutation to trifluorothymidine resistance was performed using the dose levels described hereafter:

- experiment 1: 3-hour treatment, without S9: 70.0, 53.8, 41.4, 31.9, 24.5, 18.9 and 14.5 µg/mL

- experiment 1: 3-hour treatment, with S9: 140, 108, 82.8, 63.7, 49.0, 37.7 and 29.0 µg/mL

- experiment 2: 24-hour treatment, without S9: 6.3, 35.6, 27.4, 21.1, 16.2 and 12.5 µg/mL

- experiment 2: 3-hour treatment, with S9: 80.0, 66.7, 55.6, 46.3, 38.6, 32.2 and 26.8 µg/mL

- experiment 3: 3-hour treatment, without S9: 50.0, 33.3, 22.2, 14.8, 9.90, 6.60 and 4.40 µg/mL

 In the first experiment, in the absence of S9 metabolic activation, insufficient number of cells survived to treatment at the highest dose level tested (70.0 µg/mL). Severe toxicity reducing relative total growth (%RTG) to 1% was noted at 53.8 µg/mL. The next lower dose level selected (41.4 µg/mL) yielded mild toxicity reducing RTG to 46% of the concurrent negative control value. No relevant toxicity was noted over the remaining dose levels tested.

In the presence of S9 metabolic activation, insufficient number of cells survived to treatment at the two highest dose levels (140 and 108 µg/mL). Severe toxicity reducing RTG to 7% of the concurrent negative control value was observed at 82.8 µg/mL. Dose-related toxicity was noted over the remaining dose levels tested.

 

In the second experiment, in the absence of S9 metabolic activation using a long treatment time, no or insufficient number of cells survived to treatment at the two highest dose levels (46.3 and 35.6 µg/mL). The next lower dose level selected (27.4 µg/mL) yielded severe toxicity reducing RTG to 3% of the concurrent negative control value. Marked toxicity reducing RTG in a range of 10-28% was seen in the remaining dose levels tested (21.1, 16.2 and 12.5 µg/mL). In the presence of S9 metabolism, severe toxicity reducing RTG to 8% of the concurrent negative control value was noted at the highest dose level (80.0 µg/mL). Dose-related toxicity was noted over the remaining dose levels tested.

In the third experiment, using the long treatment time in the absence of S9 metabolic activation, an insufficient number of cells recovered to treatment at the highest dose level tested (50.0 µg/mL). The next lower dose level of 33.3 µg/mL yielded moderate toxicity reducing RTG to 40%, while slight toxicity was noted at 22.2 and 14.8 µg/mL reducing RTG to 68% and 76%, respectively. No toxicity was observed over the remaining dose levels. Despite the steep decline of relative total growth induced by the test item and the resulting difficulty to select the appropriate dose levels, concentrations assayed in the second and third experiment in the absence of S9 metabolism covered a range from the maximum to no toxicity and were considered adequate to evaluate the potential mutagenicity of the test item.

In the absence or presence of S9 metabolic activation, no statistically increases in mutant frequency were observed at any treatment time. It was concluded that tert-pentyl-2 -ethylhexanoate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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
Target gene:
Histidine operon
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
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.
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
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
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'.

Results: see attached doc

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 March to 6 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline; coherence between data, results and conclusions
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 IU/ml + Penicillin G 50.000 units/ml
F 68 Pluronic

Minimal medium B

RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 units/ml + Penicillin G 50.000 units/ml

Complete medium (5%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium (10%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium A (20%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium B (20%)

Minimal medium B
Horse serum (heat-inactivated)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix:
70.0, 53.8, 41.4, 31.9, 24.5, 18.9 and 14.5 µg/mL for the first experiment (3-hour treatment)
46.3, 35.6, 27.4, 21.1, 16.2 and 12.5 µg/mL for the second experiment (24-hour treatment)
50.0, 33.3, 22.2, 14.8, 9.90, 6.60 and 4.40 µg/mL for the third experiment (24-hour treatment)

Experiments with S9 mix:
140, 108, 82.8, 63.7, 49.0, 37.7 and 29.0 µg/mL for the first experiment (3-hour treatment)
80.0, 66.7, 55.6, 46.3, 38.6, 32.2 and 26.8 µg/mL for the second experiment (3-hour treatment)



Vehicle / solvent:
Vehicle used: dimethylsulfoxide (DMSO)

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: fluctuation method in medium

DURATION
- Exposure duration: 3 and 24 hours in the absence of S9 metabolism; 3 hours in the presence of S9 metabolis
- Expression time (cells in growth medium): Two days after treatment (48 hours)
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF CELLS EVALUATED: 1.6 cells/well plated in each of 96-well plates (two)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

DETERMINATION OF MUTATION
- Method: Induced mutant frequency (IMF)
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:

(i) The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell
method (126 x 10-6) at one or more doses.

(ii) There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive
responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological
significance. Any increase in mutant frequency should lie outside the historical control range to have biological
relevance.

At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder
effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and
not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is
generally recommended that such data are treated with caution or excluded from consideration.
Statistics:
Dunnett test. Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No precipitate was noted upon addition of the test item to the cultures in all treatment series and by the end of treatment incubation period.
The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

RANGE-FINDING/SCREENING STUDIES (Cytotoxicity test):
On the basisi of solubility results, a citotoxicity assay was performed in the absence and presence of S9 metabolic activation, at a maximum dose level of 2300 µg/mL and at a wide range of lower dose levels: 1150, 575, 288, 144, 71.9, 35.9, 18.0 and 8.98 µg/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived to treatment at the five highest dose levels tested . At the next lower dose level (71.9 µg/mL) severe toxicity was observed reducing relative survival (RS) to 2% of the concurrent negative control value, while relevant toxicity was noted over the remaining dose levels. Using the 24 hour treatment time, no cells survived to treatment at the six highest dose levels. Test item treatment at 35.9 µg/mL yielded moderate toxicity reducing RS to 21%. No toxicity was observed over the remaining dose levels tested.
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no or insufficient number of cells survived to treatment at the five highest dose levels. No relevant toxicity was observed over the remaining dose levels tested.


COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive control treatments were included in the mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

ADDITIONAL INFORMATION ON MUTAGENICITY:
Based on the cytotoxicity results obtained in the preliminary assay, three independent assays for mutation to trifluorothymidine resistance were performed using the dose levels described in the following table:

First experiment:
70.0, 53.8, 41.4, 31.9, 24.5, 18.9 and 14.5 ug/mL (3-hour treatment time in the absence of S9 mix)
140, 108, 82.8, 63.7, 49.0, 37.7 and 29.0 ug/mL (3-hour treatment time in the presence of S9 mix)

Second experiment:
46.3, 35.6, 27.4, 21.1, 16.2 and 12.5 ug/mL (24-hour treatment time in the absence of S9 mix)
78.0, 60.0, 46.2, 35.5, 27.3, 21.0 and 16.2 ug/mL (3-hour treatment time in the presence of S9 mix)

Third experiment:
50.0, 33.3, 22.2, 14.8, 9.90, 6.60 and 4.40 ug/mL (24-hour treatment time in the absence of S9 mix)

In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time.
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that TERT-PENTYL 2-ETHYLPEROXYHEXANOATE does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions
Executive summary:

The test item, tert-pentyl-2 -ethylhexanoate, was examined for mutagenic activity by assaying for the induction of 5‑trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

The study was designed to comply with the  OECD 476 guideline (adopted July 1997) and was in compliance with GLP.

Methods

The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.

The dose levels for the positive controls were:

-Without S9 mix:methylmethanesulphonate (MMS) 10.0 ug/mL (3 -hour treatment time); 5.00 ug/mL (24 -hour treatment time)

-With S9 mix:benzo(a)pyrene (B(a)P) 2.00 ug/mL

A cell suspension (1 x 106cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.

The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS). Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained without metabolic activation, the second experiment in the absence of S9 metabolism was performed using a longer treatment time (24 hours).

In the second experiment in the absence of S9 metabolism (24 hour treatment), an excessive and unexpected toxicity was noted at the dose levels selected for treatment and no sufficient concentrations were analysable for the mutation induction. A third experiment was then performed. A dose range from 2.38 to 26.0 µg/mL was selected on the basis of the toxicity results observed in the previous experiment. However, due to a technical oversight during the formulation of the test item, the dose levels tested were 50.0, 33.3, 22.2, 14.8, 9.90, 6.60 and 4.40 µg/mL.

After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2 x 105cells/mL.

The cultures were incubated at 37°C in a 5% CO2atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Plating for 5-trifluorothymidine resistance

After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 μg/ml) and an estimated 2 x 103cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

Plating for viability

After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Results

A main assay for mutation to trifluorothymidine resistance was performed using the dose levels described in the following table:

 Experiment no  S9 Treatment time (h)  Dose levels (ug/mL)
 1  -  3  70.0, 53.8, 41.4, 31.9, 24.5, 18.9 and 14.5
 1  +  3  140, 108, 82.8, 63.7, 49.0, 37.7 and 29.0
 2  -  24  46.3, 35.6, 27.4, 21.1, 16.2 and 12.5

 2

3

 +

-

 3

24

80.0, 66.7, 55.6, 46.3, 38.6, 32.2 and 26.8

50.0, 33.3, 22.2, 14.8, 9.90, 6.60 and 4.40

Solvent and positive control cultures were included in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range (50-200 x 10-6viable cells). The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). The cloning efficiencies at Day 2 in the negative control cultures and the control growth factor over 2 days fell within the range.The study was accepted as valid.

Survival after treatment

In the first experiment, in the absence of S9 metabolic activation, insufficient number of cells survived to treatment at the highest dose level tested (70.0 µg/mL). Severe toxicity reducing relative total growth (%RTG) to 1% was noted at 53.8 µg/mL. The next lower dose level selected (41.4 µg/mL) yielded mild toxicity reducing RTG to 46% of the concurrent negative control value. No relevant toxicity was noted over the remaining dose levels tested.

In the presence of S9 metabolic activation, insufficient number of cells survived to treatment at the two highest dose levels (140 and 108 µg/mL). Severe toxicity reducing RTG to 7% of the concurrent negative control value was observed at 82.8 µg/mL. Dose-related toxicity was noted over the remaining dose levels tested.

In the second experiment, in the absence of S9 metabolic activation using a long treatment time, no or insufficient number of cells survived to treatment at the two highest dose levels (46.3 and 35.6 µg/mL). The next lower dose level selected (27.4 µg/mL) yielded severe toxicity reducing RTG to 3% of the concurrent negative control value. Marked toxicity reducing RTG in a range of 10-28% was seen in the remaining dose levels tested (21.1, 16.2 and 12.5 µg/mL). In the presence of S9 metabolism, severe toxicity reducing RTG to 8% of the concurrent negative control value was noted at the highest dose level (80.0 µg/mL). Dose-related toxicity was noted over the remaining dose levels tested.

In the third experiment, using the long treatment time in the absence of S9 metabolic activation, an insufficient number of cells recovered to treatment at the highest dose level tested (50.0 µg/mL). The next lower dose level of 33.3 µg/mL yielded moderate toxicity reducing RTG to 40%, while slight toxicity was noted at 22.2 and 14.8 µg/mL reducing RTG to 68% and 76%, respectively. No toxicity was observed over the remaining dose levels. Despite the steep decline of relative total growth induced by the test item and the resulting difficulty to select the appropriate dose levels, concentrations assayed in the second and third experiment in the absence of S9 metabolism covered a range from the maximum to no toxicity and were considered adequate to evaluate the potential mutagenicity of the test item.

At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration. Accordingly, we have excluded from the statistical analyses, mutation data obtained in the absence of S9 metabolism at 53.8 µg/mL (first experiment) and 27.4 µg/mL (second experiment) and in the presence of S9 metabolism at 82.8 µg/mL and 80.0 µg/mL in the first and second experiment, respectively.

Mutation results

In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time.

In the original second main assay, in the absence of S9 metabolism, a linear trend was indicated (p<5%) but no statistically significant increase in mutant frequency was observed at any dose level tested. Hence, the observed linear trend was considered to be attributable to a chance event not related to the action of the test item and of no biological significance.

Conclusion

It is concluded that TERT-PENTYL 2-ETHYLPEROXYHEXANOATE does not induce mutation at the TK locus of L5178Y mouse lymphoma cellsin vitroin the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test

t-amyl 2-ethylhexaneperoxoate was tested in a GLP compliant Mammalian Erythrocyte Micronucleus Test, according to OECD n° 474 guideline. Groups of 5 males and 5 females Swiss Ico:OF1 (IOPS Caw) mouse were treated by gavage with t-amyl 2 -ethylhexaneperoxoate (purity 96.5%) at doses of 0, 500, 1000, and 2000 mg/kg. One group of 5 males and 5 females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

Bone marrow cells were harvested at 24 and/or 48 hours post-treatment. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

At 2000 mg/kg/day, dyspnea, hypoactivity, sedation, staggering gait and/or pi1oerection were noted in both males and females. At 24 hours following the second treatment only piloerection persisted in males. There were no effects on MPE as wall as on PE/NE compared to the the vehicle control group. In addition the mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.

Therefore ter-amyl-2-ethylhexaneperoxoated did not induce an increase in micronucleus mouse bone marrow when tested to 2000 mg/kg (oral route).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: GLP, OECD study 474 (July 1997)
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Iffa Crédo, I'Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: individually in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum A04 C pelleted maintenance diet (SSNIFF Spezialdiät GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum drinking water filtered by a 0.22µ membrane
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 30 to 70
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Volume: 10 ml/kg
Duration of treatment / exposure:
2 administrations separated by 24 hours.
Frequency of treatment:
once daily for 2 days
Post exposure period:
24 hours
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): commomly used in this test and recommanded by the OECD guideline
- Route of administration: oral
- Frequency: one administration only
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
Femurs of the animals were removed and all the bone marrow was flushed out using fetal calf serum.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square
test value (Lovell and coll., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the x2 value (Lovell and coll., 1989).
When there was significant within-group heterogeneity, then that group was compared with the
control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969). The student 11t11 test was used for the PEINE ratio comparison.
Probability values of p0.05 was considered as significant.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
systemic toxicity
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (6 animals)
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. Hypoactivity and/or dyspnea and sometimes staggering gait were noted in all animals during at least the 4 hours following treatment. Clinical signs and any mortality were recorded for a period of 48 hours. At the end of this period, the animals were killed by C02 inhalation in excess. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no mortality was noted, the top dose-level selected for the main test was 2000 mg/kg/day.
Conclusions:
Interpretation of results (migrated information): negative
In conclusion, t-amyl 2-Ethylhexaneperoxoate did not induce an increase in micronucleus mouse bone marrow when tested to 2000 mg/kg.
Executive summary:

T-amyl 2-Ethylhexaneperoxoate was tested in a Mammalian Erythrocyte Micronucleus Test, according to the OECD n° 474 Guideline and EC 92/69/EEC B.12 guidelines in compliance with the Principles of Good Laboratory Practice.

In a bone marrow micronucleus assay, groups of 5 males and 5 females Swiss Ico:OF1 (IOPS Caw) mouse were treated by gavage with t-amyl 2-Ethylhexaneperoxoate (purity 96.5%) at doses of 0, 500, 1000, and 2000 mg/kg. 

One group of 5 males and 5 females received the vehicle under the same experimental conditions, and acted as control group. One group of 5 males and 5 females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

Bone marrow cells were harvested at 24 and/or 48 hours post-treatment. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

At 2000 mg/kg/day, dyspnea, hypoactivity, sedation, staggering gait and/or pi1oerection were noted in both males and females. At 24 hours following the second treatment only piloerection persisted in males.

The mean values of MPE as well as the PE/NE ratio in the groups treated with the test item were considered as equivalent to those of the vehicle control group.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.

Cyclophosphamide induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under these experimental conditions, t-amyl 2-Ethylhexaneperoxoate did not induce any noteworthy increase in the number of micronucleated with structural chromosome aberration, both with and without S9 mix, in any experiment. 

In conclusion, t-amyl 2 -ethylhexaneperoxoated did not induce an increase in micronucleus mouse bone marrow when tested to 2000 mg/kg.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Conclusion on genotoxicity:

In only one strain of the Ames test, a mutagenic effect was observed. These positive effect was not confirmed in the MLA. In addition the in vivo micronucleus test did not show any increase in micronucleus mouse bone marrow. Systemic effects were observed in the in vivo test, therefore the substance was considered as systemically available and the test is considered as adequate even if there was no PE/NE decrease. Regarding both in vitro and in vivo results, it is concluded that tert-amyl-2 ethylhexanoate does not induce genotoxic effects.


Justification for selection of genetic toxicity endpoint
Key study, Klimisch 1, in vivo data.

Short description of key information:
OECD 471: Ames positive only on TA 102 with S9 (Haadouk, 1999)
OECD 476: MLA negative (Salvador, 2012)
OECD 474: In vivo micronucleus test negative in mice (Haadouk, 1997)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to EU directive 67/548/EEC and according to EU Regulation (EC) N0. 1272/2008 (CLP), tert-amyl peroxy-2-ethylhexanoate is not classified for genotoxicity.