Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested for genetic toxicity in three in vitro tests and three in vivo test.

Two Ames tests were performed with (TBPEH), revealing controversial results. One of these tests showed no evidence for mutagenic activity. The results of the second Ames test indicated that under the conditions of the study, TBPEH caused positive responses in the presence of S9 mix only. Further, TBPEH was tested in a Mammalian Gene Mutation Test in V79 cells tested without and with metabolic activation (S9 mix). TBPEH induced biologically relevant and concentration dependent increases in mutant frequency. Therefore, TBPEH was considered mutagenic in this mammalian cell gene mutation test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-04-17 to 1996-08-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Tester strains TA 98 and TA 1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frame shift mutagens. Tester strains TA 1535 and TA 102 are reverted mutagens that cause base pair substitutions. Tester strain TA 100 is reverted by mutagens that cause both frame shift and base pair substitution mutations. Specificity of the reversion mechanism in E.coli is sensitive to base pair substitution mutations, rather than frame shift mutations.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: WP2 pKM101
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
without S9 mix: 0, 100, 333, 1000, 3333 and 5000 µg
with S9 mix: 0, 33, 100, 333, 1000 and 5000 µg
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at 500 mg/mL, the maximum concentration tested.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
For the S. typhimurium strain TA 1537, TA 1537, TA 100, TA 98 and the E.coli strain WP2 uvrA (pKM101); with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sterigmatocystin
Remarks:
For the S.typhimurium strain 102 and the E. coli WP2 (pKM101) strain; with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For the S. typhimurium strain TA 98, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For the S. typhimurium strain TA 1535, TA 100; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For the S. typhimurium strain TA 1537; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
For the S. typhimurium strain TA 102; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For the E. coli WP2 (pKM101) and WP2 uvrA (pKM101) strain; without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)


Treatment of test system
- 0.5 mL of S9 mix or Sham mix, 100 µL tester strain and 50 µL of vehicle, test article dilution or positive control will be added to 2 mL of molten selective top agar at 45 ± 2 °C. When necessary to achieve the target concentration, aliquots of other than 50 µL of test article/ vehicle/ positive control will be plated. The mixture will be vortex mixed and overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay has solidified, the plates will be inverted and incubated for approximately 48 to 72 hours at 37 ± 2 °C. Plates that are not counted immediately following the incubation period will be stored at 4 ± 2°C.

Colony Counting
- The condition of the bacterial background lawn will be evaluated for evidence of test article toxicity and precipitate. Evidence of toxicity will be scored relative to the vehicle control plate and recorded along with the revertant count for that plate.


NUMBER OF REPLICATIONS: All dose levels of test article, vehicle controls and positive controls were run in triplicate.



Evaluation criteria:
Evaluation of results:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. Statistical tests are not indicated.

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article. Data sets for strains TA 1535 and TA 1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA 98, TA 100, TA 102, WP2 uvrA (pKM101) and WP2 (pKM101) were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. Statistical tests are not indicated.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: WP2 pKM101
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In Experiment B1, the initial assay, positive responses were observed with tester strains TA100 (2.3-fold, maximum increase, TA 102 (7.8-fold maximum increase), WP uvrA (pKM101) (5.3-fold, maximum increase) and WP2 (pKM101) (20.7-fold, maximum increase) in the presence of S9 activation. No positive responses were observed in any of the remaining tester strain/activation combinations.
In Experiment B2, the independent repeat assay, positive responses were observed with tester strains TA 100 (2.0-fold, maximum increase), WP uvrA (pKM101) (3.6-fold, maximum increase) and WP2 (pKM101) (15.4-fold, maximum increase) in the presence of S9 activation. Due to possible tester strain culture contamination, tester strain TA98 in the presence and absence of S9 activation was not evaluated but was retested in Experiment B3. Due to an unacceptable vehicle control value, tester strain TA 102 in the presence of S9 activation was not evaluated but was retested in Experiment B3. No positive responses were observed in any of the remaining tester strain/activation combinations.

In Experiment B3, no positive response was observed with tester strain TA98 in the presence and absence of S9 activation. Due to an unacceptable vehicle control value, tester strain TA 102 in the presence of S9 activation was not evaluated but was retested in Experiment B4.

In Experiment B4, a positive response was observed with tester strain TA102 (3.8-fold, maximum increase) in the presence of S9 activation.
Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Salmonella/Escherichia coli Mutagenicity Assay indicate that under the conditions of this study, tert.-Butylperoxy- 2-ethylhexanoat did cause positive responses with tester strains TA100, TA102, WP2 uvrA (pKM101) and WP (pKM101) in the presence of Aroclor-induced rat liver S9.
Executive summary:

In a reverse gene mutation assay in bacteria, strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 102) and at the tryptophan locus of Escherichia coli tester strains WP2 (pKM101) and WP uvrA (pKM101) were exposed to tert.-Butylperoxy- 2-ethylhexanoat in the presence and absence of S9 activation according to OECD guideline 471. The assay was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test article.

DMSO was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at 500 mg/mL, the maximum concentration tested.

In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a stock concentration of 100 mg/mL and a 50 µL plating aliquot. Based on the findings of the toxicity assay, the maximum doses plated in the mutagenicity assay was 5000 µg per plate for all tester strains in the presence and absence of S9 activation.

In the mutagenicity assay positive responses were observed. No precipitate was observed and toxicity was generally observed at ≥ 1000 to 5000 µg per plate in the presence of S9 activation. In Experiment B1, the initial assay, positive responses were observed with tester strains TA100, TA102, WP2 uvrA and WP2 (pKM101) in the presence of S9 activation. No positive responses were observed in any of the remaining tester strain/activation. In Experiment B2, the independent repeat assay, positive responses were observed with tester strains TA100, TA102, WP2 uvrA and WP2 (pKM101) in presence of S9 activation. No positive responses were observed in any of the remaining tester strain/activation.

In Experiment B3, no positive response was observed with tester strain TA98 in presence and absence of S9 activation. In Experiment B4, a positive response was observed with tester strain TA102 in the presence of S9 activation.

Under the conditions of this study, test article tert.-Butylperoxy- 2-ethylhexanoat was concluded to be positive in the Salmonella/ Escherichia coli Mutagenicity Assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-18 to 2010-02-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
The V79 cells are exposed to the test item both with and without metabolic activation. The study was determined whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:
Experiment 1 (3-hour treatment period without and with S9 mix): 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 ug/mL
Experiment 2 (20-hour treatment period without S9 mix): 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 ug/mL
Experiment 2 (3-hour treatment period with S9 mix): 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DSMO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DSMO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
The test item, tert. butylperoxy-2-ethylhexanoate (TBPEH) was tested in a Mammalian Gene Mutation Test in V79 cells. The test item was dissolved in DMSO and the used concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).
Evaluation criteria:
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (10e6 cells: 5 plates at 2 x 10e5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10e6 cloneable cells. Assay acceptance criteria:
- The mutant frequency in the negative (solvent) control cultures is in line with the historical control data.
- The positive control chemicals induce a clear increase in mutant frequency.
- The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 0 and 70% to 130% on Day 7.
Statistics:
Wilcoxon-Mann-Whitney U-test was used to determine whether or not there are statistically significant increases in mutant frequency.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There was very clear evidence of toxicity with the test item (6-14 fold; tox. (survival) ~90% - 10%) in both absence and presence of metabolic activation (S9 mix) when compared to the solvent controls confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
The mutation frequencies of the negative and positive control cultures were consistent with the historical control data from previous studies performed at the laboratory and demonstrated validity and sensitively of the test system.
In Experiment 1, there were no toxicologically significant increases in mutant frequency at any concentration in the absence of metabolic activation. When the test item was examined in the presence of metabolic activation there were dose-associated increases in mutant frequency compared to the negative control. These increases were statistically significant at concentrations of 156.25, 234.37 and 312.50 ug/mL.

In Experiment 2, the mutant frequency of the cells showed statistically significant increases compared to the concurrent control, when the test item was examined without S9 mix over a prolonged treatment period (20 hours). The 3-hour treatment in the presence of S9 mix caused significant increases in mutant frequency, confirming the findings in Experiment 1. These increases in mutant frequency showed dose-response relationships and were statistically significant at most concentrations tested.
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.
pH and osmolality of control and treatment solution were measured. In Experiments 1 and 2 no significant differences between treatment and control groups were observed .

no remarks

Conclusions:
Tert.-Butylperoxy- 2-ethylhexanoat tested both without and with metabolic activation (S9 mix), induced increases in mutant frequency in the HPRT assay in chinese hamster lung cells. Therefore, tert.-Butylperoxy- 2-ethylhexanoat was mutagenic in this in vitro mammalian cell gene mutation test performed with V79 cells.
Executive summary:

The test item, tert.-Butylperoxy- 2-ethylhexanoat was tested in a Mammalian Gene Mutation Test in V79 cells according to EU-method B.17. The test item was dissolved in DMSO and the following concentrations were selected based on the results of the preliminary study (without and with metabolic activation) for the main study: 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 ug/mL. Two independent main experiments were performed. In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration without S9 mix. Three-hour treatment in the presence of S9 mix caused dose-related increases in mutant frequency. These increases were statistically significant at concentrations of 156.25, 234.37 and 312.50 ug/mL. In Experiment 2, the mutant frequency showed dose-associated statistically significant increases compared to the concurrent control without S9 mix over a prolonged treatment period of 20 hours. In this experiment, a 3-hour treatment in the presence of S9 mix caused statistically significant dose-related increases in mutant frequency, further supporting the findings in Experiment 1. Tert.-Butylperoxy- 2-ethylhexanoat tested both without and with metabolic activation (S9 mix), induced increases in mutant frequency in this test in Chinese hamster lung cells. Therefore, tert.-Butylperoxy- 2-ethylhexanoat was mutagenic in this in vitro mammalian cell gene mutation test performed with V79 cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test item was tested in an in vivo mouse micronucleus assay. The test item did not induce a significant or biological relevant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice up to limit dose. TBPEH was therefore concluded to be negative in the micronucleus test.

In addition, tert-butyl-2-ethylperoxyhexanoate was tested according to a special protocol as requested by ECHA for its ability to induce gene mutation in the lacZ transgene in liver, forestomach, kidney, small intestine and developing germ cells from Muta™Mice (CD2 lacZ80Ha2 strain). In this assay some indication for a mutagenic effect in somatic cells (liver) were found, which however are difficult to interpret, whereas no indication for germ cell mutations were noted following dosing for 42 days at up to 800 mg/kg bw/day. However, a second TRM assay showed that the test item did not induce gene mutation in the liver, forestomach, kidney and small intestine of transgenic mice (is negative) after an exposure time of 28 days.

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Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non GLP; Both doses by far exceeded the max. doses recommended by current OECD Guideline (474)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Both doses by far exceeded the max. doses recommended by current OECD Guideline (474)
Principles of method if other than guideline:
NA
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Males: 27 - 35 g; Females: 24 - 29.9 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet (e.g. ad libitum): ad libitum, TEKLAD Certified Rodent Chow 7012C
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23°C
- Humidity (%): 50 ± 20°C

Route of administration:
oral: gavage
Vehicle:
Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in corn oil at a concentration of 500 mg/mL, the maximum concentration tested.
Details on exposure:
NA
Duration of treatment / exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by oral gavage at a constant volume of 20 mL/kg bw. Mice were observed after dose administration for clinical signs of chemical effect.
Frequency of treatment:
24, 48, 72 hrs after dose administration
Post exposure period:
None
Dose / conc.:
20 other: mL/kg bw
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP) will be administrated as the positive control at a dose of 60 mg/kg. CP will be administrated by the same route as the test article.
Tissues and cell types examined:
The micronucleus study evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice.
Details of tissue and slide preparation:
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control (p ≤ 0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1979; Mackey and Mac Gregor, 1979)
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration (p>0.05, Kastenbaum-Bowman).

No remarks

Conclusions:
Under the conditions of the assay described in this report, tert.-Butylperoxy- 2-ethylhexanoat did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The test article, tert.-Butylperoxy- 2-ethylhexanoat, was tested in the mouse micronucleus assay. In the study, test and control articles were administered in a constant volume of 20 mL/kg bw by a single oral gavage.

Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in corn oil at a concentration of 500 mg/mL, the maximum concentration tested.

In the pilot assay (toxicity test), male mice were dosed with 1, 10, 100 or 1000 mg test article/kg bw and male and female mice were dosed with 5000 mg/kg bw. Mortality occurred an 1/5 males and 3/5 females at 5000 mg/kg. Clinical signs observed after dose administration included: lethargy in male mice at 1000 mg/kg and in male and female mice at 5000 mg/kg, prostration and crusty eyes in one male mouse and diarrhoea in one female mouse at 5000 mg/kg. Treatment-related mortality was observed to be less than 50 % in the pilot toxicity study. However, in females the mortality was estimated to be 60% at 5000 mg/kg. Therefore, the high dose in the micronucleus study for males was set at 5000 mg/kg. For females the high dose was set at 4000 mg/kg which was estimated to be approximately 80 % of 5000 mg/kg. Both doses by far exceeded the maximum doses recommended by current OECD guideline.

In the micronucleus assay, male mice were dosed with 1250, 2500 or 5000 mg/kg and female with 1000, 2000 or 4000 mg/kg bw of tert.-Butylperoxy- 2-ethylhexanoat. Mortality occurred in 1 /20 male mice at 5000 mg/kg and 2 /25 female mice at 4000 mg/kg. Clinical signs following dose administration included: lethargy in male and female mice at all dose levels, diarrhoea in male mice at 2500 and 5000 mg/kg and in female mice at 2000 and 4000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Moderate reductions (up to 36 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the vehicle control groups. A reduction of 36 % suggests bioavailability of the test article to the target organ, the bone marrow.

Under the conditions of the assay described in this report, tert.-Butylperoxy- 2-ethylhexanoat did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow even at pronounced systemically toxic doses and was concluded to be negative in the micronucleus test using male and female ICR mice.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-09-07 and
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD TG 488 (2013)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
transgenic rodent mutagenicity assay
Species:
mouse
Strain:
other: CD2-LacZ80/HazfBR (Muta Mouse)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan Laboratory Animals, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 25.8 to 28.7 g
- Assigned to test groups randomly: yes, under following basis: body weights on day 1 (weight range of animals used was within +/- 20% of mean weight)
- Fasting period before study: none
- Housing: in groups of two or three animals per cage
- Diet: ad libitum (pellet diet CRF-1, Oriental Yeast)
- Water: ad libitum (tap water from water bottles)
- Acclimation period: 7 days (day -8 to day 1)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8 to 23.2
- Humidity (%): 43.6 to 54.6
- Air changes (per hr): 12 times or more
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: standard vehicle, good stability of test substance in vehicle
- Concentration of test material in vehicle: 10, 30 and 80 mg/mL
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
2.4 g of the test substance were weighed into a graduated glass bottle. Corn oil was poured into the bottle to make a total volume of 30 mL, and a stock formulation of 80 mg/mL was prepared. The stock solution was diluted with an appropriate volume of corn oil to make a 30 mg/mL formuation. 10 mg/mL formulation was prepared by diluting the 30 mg/mL formulation in corn oil. The test substance formulations was divided into 2 to 4 portions (including 1 portion as reserve) for each administration and stored in a refrigerator (acceptable range: 2 to 8°C) until use. Before dividing, the test substance formulations was collected for the concentration analysis only in the first preparation.

STABILITY
The test substance formulations in corn oil at 2 and 500 mg/mL were stable for 24 hours when stored at 15 to 25°C, and for 3 days when stirred at 2 to 8°C. Sufficient homogeneity had also been determined and confirmed in previously studies (28-days study, report no. SL-LT-028/10 and validation of a determination method in corn oil, report no. G306 (673-001))
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
once daily
Post exposure period:
3 days
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 males (5 animals were evaluated)
Control animals:
yes, concurrent vehicle
Positive control(s):
N-ethyl-N-nitrosourea (ENU)
- Justification for choice of positive control(s): according to guideline
- Route of administration: intraperitoneally
- Doses / concentrations: 100 mg/kg/day
- Frequency/Duration: once daily for 2 consecutive days
Tissues and cell types examined:
Liver, stomach, kidney, small intestine
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of the 14-day range finding test.

SAMPLING TIMES:
The organs were removed 3 days after last dosing.

TISSUE PREPARATION AND DNA EXTRACTION:
DNA was extracted from liver, forestomach, kidney and small intestine. Before DNA extraction organs were removed and stored as followed:
- Liver: About two samples were prepared from the left lateral lobe using a biobsy trephine (BP-50F, Kai). The samples were separately placed in microtubes and frozen in liquid nitrogen (LN2). The other parts and lobes were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
- Forestomach: The greater curvature of the stomach was incised. The stomach contents were removed by washing with physiological saline. After the observation of the mucosa (potential findings were recorded), stomach was divided into forestomach and glandular stomach. Forestomach was put into a storage bag and frozen in LN2. Glandular stomach was discarded.
- Kidney: The capsule of left kidney was removed and sliced in a thickness of approximately 1 to 2 mm (approximately 2 horizontal pieces in total). Each slice was separately placed in microtubes and frozen in LN2. The other parts and right kidney were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
- Small intestine: The contents were removed by washing with physiological saline. Small intestine was put into a storage bag and frozen in LN2.

For extraction of genomic DNA, three milliliters of the buffer for tissue breakage (containing RNase) were poured into a Dounce-type homogenizer and cooled with ice. Each frozen tissue sample was put into the homogenizer and homogenized with a pestle.
The homogenized tissue fragments were poured gently into an ice-cooled 15-mL centrifuge tube containing 3 mL of 0.5 mol/L sucrose solution, and centrifuged (centrifuge LC-122, Tomy Seiko) at 3000 rpm (1710 G) for 10 minutes. The supernatant was removed with a dropper and 3 mL of cooled RNase-containing Dounce buffer were added to the tube and mixed well (nuclear/cell suspension). Then, 3 mL of proteinase K solution were added to the nuclear/cell suspension and gently mixed by inversion. This suspension was incubated at 50°C for about 3 hours until it became clear. The same volume (6 mL) of Ph/Cl mixture was added to the solution and mixed by inversion a few times, mixed by using a rotator for 10 minutes, and centrifuged (centrifuge LC-122) at 2500 rpm (1190 G) for 10 minutes. Next, the upper layer (water layer) was gently collected and transferred into another 15-mL centrifuge tube by a transfer pipette. This procedure was repeated twice (volume of Ph/Cl mixture was same as the removed water layer). After removal of the water layer, the same volume of chloroform/isoamyl alcohol mixture was poured into the tube. The contents were mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rpm for 10 minutes. Then, the water layer was transferred into another 50-mL centrifuge tube. Genomic DNA was extracted by gradually adding ethanol in the tube. Extracted genomic DNA was transferred into a microtube containing 70% ethanol and stood for about 10 minutes. The contents were centrifuged (centrifuge MX-160) at 13000 rpm (13240 G) for 10 minutes. After the supernatant was removed as much as possible using a micropipette, the tube was stood at room temperature to evaporate ethanol. An appropriate volume (50 to 150 μL) of TE buffer (lot No. 02234G, Nippon Gene) was added to the tube. The tube was stood overnight at room temperature to dissolve DNA residues. The DNA solution was stored in a refrigerator after preparation. The concentration of DNA in the genomic DNA solution was measured using a spectrophotometer (NanoDrop® ND-1000, AGC TECHNO GLASS) and adjusted to about 100 to 600 μg/mL with the TE buffer.

DNA PACKAGING:
One red tube of Transpack packaging extract was thawed. Using a pipette, 10 μL of genomic DNA solution adjusted to a concentration of about 100 to 600 μg/mL were transferred to the red tube. The packaging reaction was mixed by pipetting and the tube was incubated at 30°C for 90 minutes. Next, a blue tube of Transpack packaging extract was thawed, and 10 μL of it were transferred to the red tube containing a packaging reaction, and
mixed in the same manner. It was incubated at 30°C for another 90 minutes, and 700 μL of SM buffer were added to the tube, and mixed (packaged DNA sample). The packaged DNA sample was stored on ice until use.

PLATING OF PACKAGING DNA:
One milliliter of E. coli suspension for calculating total number of plaques (for tittering) and 2 mL for calculating mutant frequency (for selection) will be dispensed into each tube. Then, the entire volume of packaged DNA sample (about 700 μL) will be added to E. coli suspension in the tube for selection (total of about 2700 μL) and mixed. The tube will be incubated at room temperature for about 20 to 30 minutes to allow phage to infect E. coli. Thirty microliters of the content will be diluted 10-fold with 270 μL of LB broth containing 10 mmol/L magnesium sulfate. Thirty microliters of it will be transferred to the tube for tittering and stirred. The magnesium sulfate solution (1 mol/L) will be added to the LB top agar at a volume ratio of 1:100 to 2:100 to make the top agar for tittering. The P-gal solution will be added to the LB top agar at a volume ratio of 1:100 to 2:100 to make the top agar for
selection. Then, 17 mL of top agar containing magnesium sulfate will be added to the tube for tittering and mixed. The contents will be poured over a LB agar plate. To the tube for selection, 16 mL of top agar containing P-gal will be added and the contents will be poured over a LB agar plate in the same manner as the tube for tittering. The agar plate will be incubated in an incubator at 37°C over night. The above packaging procedure or genomic DNA extraction-to-packaging procedures will be repeated until the total number of plaques per animal reached 300000.

METHOD OF ANALYSIS:
- Calculation of the total number of plaques
The number of plaques in the plates for titering (N) was counted, and then the total number of plaques was calculated using the following equation

Total number of plaques = N x (300 µL/30 µL) x (2700 µL/30 µL) = 900 x N

- Counting of mutant plaques
The number of plaques in the plates for selection was counted and those plaques were recognized as mutant plaques.

- Calculation of mutant frequency
The lacZ gene was selected as a reporter gene. The mutant frequency was calculated by dividng the number of mutant plaques by the total number of plaques.

Mutant frequency = Number of mutant plaques/Total number of plaques

- Data processing
The obtained data for in-life phase of this study were collected and analyzed using the computer system.
Evaluation criteria:
The results were evaluated as positive when the mutant frequency in the test substance-treated group was significantly different from that in the negative control group. However, final judgment was made in consideration of biological relevance under the test conditions.
Statistics:
The data on the mutant frequency from the negative control group and each test substance-treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant on Bartlett’s test), Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group. If there was no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyze the differences.

The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyze the differences.

In the additional assay, the data on the mutant frequency from the negative control group and the 800 mg/kg/day group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyze the differences.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the results of the 14-day repeated day study (1000 mg/kg/day in male and female Muta mice), post dosing observations were limited to reduced activity and semi-closed eyes, noted in all male animals on Day 6 only. There were no adverse effects on body weight. Histopathology assessments of the forestomach from these animals identified minimal or slight squamous cell hyperplasia in all animals given the test item. This was characterized by increased thickness and numbers of epithelial cells in the squamous epithelium of the forestomach, with occasional mitotic figures. Based on these results, dosages of 800, 300 and 100 mg/kg/day were selected for the main study. No substantial differences in the toxicity between sexes were observed and therefore male animals only were used in this study.

RESULTS OF DEFINITIVE STUDY
- Liver: In the negative control group, the mean ± SD of mutant frequency among the individuals was 41.1 ± 19.5 (×10E-6). The means ± SD of mutant frequencies in the test item treated groups, 100, 300 and 800 mg/kg/day, were 46.7 ± 14.1 (×10E-6), 36.1 ± 12.7 (×10E-6) and 37.9 ± 11.1 (×10E-6), respectively, and no statistically significant increase was observed compared with the negative control group. In the positive control group, the mean ± SD of mutant frequency among the individuals was 147.3 ± 60.0 (×10-6) and a statistically significant increase was observed compared with the negative control group.

- Forestomach: In the negative control group, the mean ± SD of mutant frequency among the individuals was 37.4 ± 8.4 (×10E-6). The means ± SD of mutant frequencies in the test item treated groups, 100, 300 and 800 mg/kg/day, were 46.7 ± 10.3 (×10E-6), 37.6 ± 8.5 (×10-E6) and 38.4 ± 6.2 (×10E-6), respectively, and no statistically significant increase was observed compared with the negative control group. In the positive control group, the mean ± SD of mutant frequency among the individuals was 519.2 ± 99.1 (×10E-6) and a statistically significant increase was observed compared with the negative control group.

- Kidney: In the negative control group, the mean ± SD of mutant frequency among the individuals was 35.1 ± 6.3 (×10E-6). The means±SD of mutant frequencies in the test item treated groups, 100, 300 and 800 mg/kg/day, were 60.1 ± 20.5 (×10E-6), 40.0 ± 9.4 (×10E-6) and 44.0 ± 6.7 (×10E-6), respectively, and a statistically significant increase was observed only in the 100 mg/kg/day group (the lowest dose) as compared with the negative control group. In the positive control group, the mean ± SD of mutant frequency among the individuals was 87.7 ± 17.0 (×10E-6) and a statistically significant increase was observed compared with the negative control group.

- Small intestine: In the negative control group, the mean ± SD of mutant frequency among the individuals was 53.3 ± 13.5 (×10E-6). The means ± SD of mutant frequencies in the test item treated groups, 100, 300 and 800 mg/kg/day, were 64.6 ± 12.4 (×10E-6), 58.6 ± 6.8 (×10E-6) and 83.3 ± 24.6 (×10E-6), respectively, and a statistically significant increase was observed in the 800 mg/kg/day group as compared with the negative control group.
In the positive control group, the mean ± SD of mutant frequency among the individuals was 783.3 ± 134.0 (×10E-6) and a statistically significant increase was observed compared with the negative control group.

- Body weight and general conditions: No changes in body weights or in the general condition were observed in any of the test substance treated groups.

- Organ weight: In the 800 mg/kg/day treated group, the relative liver weight was about 0.7% higher than that in the negative control group. There was no difference in the absolute or relative kidney weight of the test substance treated groups compared with that of the negative control group.

- Gross necropsy: There were no macroscopic findings related to test item treatment in the liver, forestomach, small intestine or kidney.

Analysis of test substance formulations

The determined concentrations of TBPEH were 10.28, 31.10 and 82.29 mg/mL for the 10 mg/mL, 30 mg/mL and 80 mg/mL formulations, respectively. These values were 102.8, 103.7 and 102.9% of the nominal concentrations, satisfying the criterion, 85 to 115%. Furthermore, the corn oil sample (negative control) showed no interference peak. These results showed that the test substance formulations had been appropriately prepared and the negative control had not been contaminated by the test substance.

Additional assay

In the negative control group, the mean ± SD of mutant frequency among the individuals was 56.5 ± 17.5 (×10E-6). The mean ± SD of mutant frequencies in the test item treated group, 800 mg/kg/day, was 50.6 ± 10.5 (×10E-6), and no statistically significant increase was observed compared with the negative control group.

Conclusions:
The test item did not induce gene mutation in the liver, forestomach, kidney and small intestine of transgenic mice (is negative) under the conditions in this study.
Executive summary:

A gene mutation assay with transgenic mice (MutaTM mouse) according to OECD TG 488 (2013) was conducted to assess the potential of the test item to induce gene mutation (reporter gene: lacZ) in the liver, forestomach, kidney and small intestine. Based on the toxic effects noted in the 14-day repeated dose study (1000 mg/kg/day in male and female MutaTM mice) conducted by the Sponsor, a dosage of 800 mg/kg/day was selected as the high dose and 2 lower dosage levels of 100 and 300 mg/kg/day were also selected for the gene mutation assay. No substantial differences in toxicity between sexes were observed and therefore male animals only were used in this study. The test item was orally administered to the male transgenic mice once a day for 28 consecutive days and after 3 days of the manifestation period, the liver, forestomach, kidney and small intestine were removed and mutant frequencies were determined as described in the most recent OECD TG 488. As a result, the mutant frequencies in the liver and forestomach of all groups treated with test item did not show any statistically significant increases as compared with that of the negative control group. In the kidney, a statistically significant increase was observed in 100 mg/kg/day group as compared with the negative control group. However, the increase was observed only at the lowest dose and did not reveal any dose-response relationship. In addition, the increase was small, namely the mutant frequency in the 100 mg/kg/day group was less than two-fold that in the negative control group and inside the distribution (mean±3SD and even mean±2SD) of the historical data of the negative control group. This increase was considered to be an incidental phenomenon. In the small intestine, a statistically significant increase was observed in 800 mg/kg/day group as compared with the negative control group. However, the increase was small, namely the mutant frequency in the 800 mg/kg/day group was less than two-fold that in the negative control group and inside the distribution (mean±3SD) of the historical data of the negative control group. Therefore, the response in the small intestine in the first assay was judged to be neither clearly negative nor clear positive and an additional assay (lacZ assay included extraction of genomic DNA) was conducted in the negative control and 800 mg/kg/day groups to determine whether this response was an incidental phenomenon. In the additional assay, no statistically significant increase was observed in 800 mg/kg/day group as compared with the negative control group. Therefore, the increase in the 800 mg/kg/day group was considered to be an incidental phenomenon because reproducibility was not observed in the additional assay and the response for both assays was within the distribution (mean±3SD) of the historical control data. The mutant frequencies in the liver of the positive control group, which was treated with N-ethyl-N-nitrosourea (ENU, dosage level of 100 mg/kg/day), increased and these increases were statistically significant (p≤0.05) compared with that of the negative control group. In addition, the effects noted are in the range of the historical control data (Min: 88.7, Max: 285.8) for this positive control and demonstrates the sensitivity of the assay.

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

Additional information

The genetic toxicity of tert-butyl 2-ethylperoxyhexanoate was tested in three in vitro tests and three in vivo test.

In vitro

The three in vitro tests were two bacterial reverse mutation assay (Ames-test) according to EU method B.13/14 and OECD guideline 471 and one mammalian cell gene mutation test (HPRT assay) according to EU Method B.17 and OECD Guideline 476.

The Ames tests revealed controversial results. In one of the Ames tests there was no evidence for mutagenic activity in four histidine-dependent auxotroph of Salmonella typhimurium, strains TA 98, TA100, TA 1535 and 1537 with and without metabolic activation, using pour-plate assays. However, in the second Ames test, using several strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 102) and Escherichia coli tester strains WP2 (pKM101) and WP uvrA (pKM101) treated with and without metabolic activation system (S9-mix) S9, tert-butyl 2 ethylperoxyhexanoate revealed several positive responses in the presence of S9 mix, only.

Furthermore, tert-butyl 2-ethylperoxyhexanoate was tested in a Mammalian Gene Mutation Test in V79 cells following EU-method B.17 and OECD guideline 476. tert-butyl 2-ethylperoxyhexanoate tested both without and with metabolic activation (S9 mix) induced biologically relevant and concentration dependent increases in mutant frequency. Therefore, tert-butyl 2-ethylperoxyhexanoate was mutagenic in this in vitro mammalian cell gene mutation test.

In vivo

Mouse micronucleus assay

The available in vivo mouse micronucleus assay with tert-butyl 2-ethylperoxyhexanoate did not reveal any indication for a potential to cause chromosal mutagenic effects The test item tested up to limit doses did not induce any relevant or significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. TBPEH was therefore concluded to be negative in this micronucleus test in male and female ICR mice.

TRM assay

Based on the request from ECHA, tert-butyl-2-ethylperoxyhexanoate was tested for its ability to induce gene mutation in the lacZ transgene in liver, forestomach, kidney, small intestine and developing germ cells from Muta™Mice (CD2 lacZ80Ha2 strain) after a prolonged administration time of 42 days. Males (7 per group) only were used, as no gender differences observed in the Range Finder Experiment. Doses (100, 300 and 800 mg/kg bw/day) were selected on the basis of post dose observations and histopathology findings observed in a 14 day Range-Finder Experiment at 1000 mg/kg/day in male and female Muta™Mice. Test article and vehicle control were administered orally via gavage daily for 42 consecutive days. Positive control (Ethylnitrosurea (ENU)) was administered daily for 28 consecutive days. No post-dose observations related to dosing with tert-butyl-2-ethylperoxyhexanoate were seen. The liver, forestomach, kidney, small intestine and developing germ cells were taken from all test article and vehicle control treated animals on Day 45 (i.e. 3 days after final test article addition), and from positive control animals on Day 31 (i.e. 3 days after the final test article addition). These tissues were analysed for mutation. All individual packaging reactions resulted in at least 30,000 pfu and at least 1 mutant plaque. For all animals data were generated for at least 200,000 pfu per tissue, from at least three independent packaging reactions. At least 1 million pfu were obtained per group, per tissue from a minimum of five animals. In liver, forestomach, kidney and small intestine, the concurrent positive control induced a significant increase mutant frequency (MF) compared to the vehicle control animals. For developing germ cells, tissue match positive control DNA had a three-fold higher MF than the study vehicle control samples, thus confirming a positive response in developing germ cell DNA could be detected. The assay was therefore accepted as valid. Statistically significant increases compared to the concurrent control in mutant frequency (MF) were observed in the liver following dosing with tert-butyl-2-ethylperoxyhexanoate at 300 and 800 mg/kg bw/day, revealing also some dose response relationship. The majority of individual animal MF were also elevated compared to the concurrent vehicle control. Overall, however only in one animal the range of laboratory’s historical control data was exceeded for the liver taken into account that this data reflect a 28 day treatment period only and no data at all exist for a prolonged treatment of 42 days. In addition, a small but statistically significant increase in MF was observed in the forestomach following dosing with tert-butyl-2-ethylperoxyhexanoate at 800 mg/kg bw/day. However, only three animals exceeded the MF of the concurrent vehicle control and all animals fell well within the very limited laboratory’s historical control data for this tissue. A biological relevance of this variation in the forestomach following tert-butyl-2-ethylperoxyhexanoate treatment although becoming statistically significant is very highly questionable taken into account the very limited set of control data and the huge variation noted in the laboratory’s historical control data for this and other organs. No statistically significant increases in MF were observed in the two other slow and fast dividing somatic cells (kidney and small intestine). Most important no effects at all were observed for the developing germ cells, for which the chosen treatment period is considered appropriate based on the recommendation in the OECD guideline. Based on this study, a clear conclusion can be drawn with regard to germ cell mutation, where no effects at all were noted at a relevant dose regimen. Thus, the test item is not a germ cell mutagen.  

The effects noted in the somatic cells are difficult to interpret. Therefore, a second study according to the current OECD guideline 488 (2013) considering a 28-day dose regimen recommended for somatic tissues was performed at a laboratory offering a reliable historical control data base for the relevant tissues. The Mouse strain (MutaMouse, reporter gene: lacZ) and dose levels used as well as the tissues examined are the same as in the first study except for the developing germ cells, which have already been assessed negative after the appropriate exposure time for those cells. The test item was orally administered to the male transgenic mice once a day for 28 consecutive days and after 3 days of the manifestation period, the liver, forestomach, kidney and small intestine were removed and mutant frequencies were determined as described in the most recent OECD TG 488. As a result, the mutant frequencies in the liver and forestomach of all groups treated with test item did not show any statistically significant increases as compared with that of the negative control group. In the kidney, a statistically significant increase was observed in 100 mg/kg/day group as compared with the negative control group. However, the increase was observed only at the lowest dose and did not reveal any dose-response relationship. In addition, the increase was small, namely the mutant frequency in the 100 mg/kg/day group was less than two-fold that in the negative control group and inside the distribution (mean±3SD and even mean±2SD) of the historical data of the negative control group. This increase was considered to be an incidental phenomenon. In the small intestine, a statistically significant increase was observed in 800 mg/kg/day group as compared with the negative control group. However, the increase was small, namely the mutant frequency in the 800 mg/kg/day group was less than two-fold that in the negative control group and inside the distribution (mean±3SD) of the historical data of the negative control group. Therefore, the response in the small intestine in the first assay was judged to be neither clearly negative nor clear positive and an additional assay (lacZ assay included extraction of genomic DNA) was conducted in the negative control and 800 mg/kg/day groups to determine whether this response was an incidental phenomenon. In the additional assay, no statistically significant increase was observed in 800 mg/kg/day group as compared with the negative control group. Therefore, the increase in the 800 mg/kg/day group was considered to be an incidental phenomenon because reproducibility was not observed in the additional assay and the response for both assays was within the distribution (mean±3SD) of the historical control data. The mutant frequencies in the liver of the positive control group, which was treated with N-ethyl-N-nitrosourea (ENU, dosage level of 100 mg/kg/day), increased and these increases were statistically significant (p≤0.05) compared with that of the negative control group. In addition, the effects noted are in the range of the historical control data (Min: 88.7, Max: 285.8) for this positive control and demonstrates the sensitivity of the assay.   Based on the results for the somatic tissues in the second study, the results of the somatic cells in the first study are considered not reliable due to the following reasons:  

Generally, a mutagenic potential should be evaluated in a tiered approach considering a 28-day administration period for somatic tissues at first. In case of a positive result a second study with a prolonged administration period for the evaluation of gene mutation induction in developing germ cells should be conducted. In contrast to these recommendations made in the OECD guideline (2013), ECHA requested instantaneously a 42-day dose regimen - also for the evaluation of the somatic tissues. The request from ECHA with regard to treatment period and tissues to be analysed was considered to be non-disputable as being part of a final decision by ECHA. However, no historical control data are available for the test regimen (42 days treatment) as requested by ECHA, but this test shows even in the limited historical control range (for 28 days) already a huge variations and only one animal of the high dose group exceeds slightly this “inappropriate” data set. Moreover, the range of historical control data at 28 day can be considered as a “worst case” as variation increases with extension of administration period and an increase of the observed range is therefore expected. For study types revealing such huge biological variation a proper historical control data set is vital for a scientific sound interpretation of variations noted. This evaluation and the importance of a proper historical control data set to allow a scientific sound evaluation is further supported by the effects (variations) noted for the small intestine. In addition, the test item did not reveal any indication for even a slight toxicity in the liver (weight effects, macroscopically and histological investigations) even tested for repeated application up to 1000 mg/kg bw/day and no effects were noted in the range finding study. However, for a gene mutation/genotoxic effect (based on the formation of reactive molecules damaging the DNA) one would expect to see damage of other essential structures in the cells/tissues (noted e.g. in histopathological investigations) together with effects on the well protected DNA.

It is therefore concluded that the test item tert-butyl-2-ethylperoxyhexanoate is neither mutagenic in somatic cells nor in germ cells in vivo and, as a consequence, not classified for mutagenicity according to Regulation (EC) 1272/2008.  




Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item does not require classification regarding genotoxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.