Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of tert-amyl hydroperoxide to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 1538 TA 98, TA 100) was evaluated according to the Ames test (direct plate incorporation assay) and in compliance with GLP (TNO, 1981). No noteworthy increase in the number of revertants was observed for all doses with and without metabolic activation on TA 1535, 1537, 1538 and 98. The significance of the slight increase in numbers of revertants with TA 100 was not clear, because at highest concentrations the toxicity of the test liquid for the bacteria interfered with the mutagenicity testing.

Tert-amyl-hydroperoxide was tested in an in vitro gene mutation assay (HPRT) using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to the "subculture method" of O'Neill and Hsie and in compliance with GLP (TNO, 1982). Tert-amyl-hydroperoxide did not induce any increase in mutant colonies and was not considered as mutagenic.

Tert-amyl hydroperoxide (purity 83.1 %) was tested in an in vitro micronucleus test in order to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells, in L5178Y TK+/-mouse lymphoma cells. The test was performed in accordance with OECD 487 guideline, and was GLP compliance. No significant increase in the frequency of micronucleated cells was noted after the 3-hour treatment in the absence of S9. Following the 24-hour treatment, an increase in the frequency of micronucleated cells (exceeding the threshold of 2.5-fold the vehicle control value) was observed at 30 µg/mL. This increase was statistically significant (p < 0.05). However, the corresponding micronucleated cells remained within the historical data range of the vehicle control (5 micronucleated cells in 1000 cells versus 0-5 for the historical data). Consequently, this increase was considered as equivocal. In all experiments in the presence of S9, slight increases in the frequency of micronucleated cells (exceeding the threshold of 2.5-fold the vehicle control value) were observed. These increases reached statistical significance in the first and third experiments and were dose-related. Therefore, these increases being reproducible in independent experiments, they were considered to be biologically significant. In conclusion, tert-amyl hydroperoxide induced chromosome damage, or damage to the cell division apparatus, in cultured L5178Y TK+/-mouse lymphoma cells, in the presence of a rat metabolizing system. In the absence of a rat metabolising system, its potential to induced chromosome damage, or damage to the cell division apparatus remained equivocal.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome aberration and aneuploidy
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 January 2012 - 06 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In vitro micronucleus)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: cryoprotective medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL in the first experiment,
.           2.5, 5, 10, 15, 20, 25, 30 and 40 µg/mL in the second experiment.

Experiments with S9 mix :
.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL for the first experiment,
.           4.7, 9.4, 18.8, 28.2, 37.5 and 75 µg/mL for the second experiment,
.           5, 10, 20, 30, 35, 40, 45 and 50 µg/mL for the third experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice: using a test item concentration of 500 mg/mL in DMSO and a treatment volume of 20 µL/2 mL culture medium, the highest recommended dose-level of 5000 µg/mL was achievable.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
see Executive summary

NUMBER OF CELLS EVALUATED: 2000/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
The biological relevance of the results should be considered first. Statistical methods are used as an aid in evaluating the test results but should not be the only determinant of a positive response. A result is considered as positive if at least a 2.5-fold increase in the number of micronucleated cells in comparison to the concurrent control is observed, with a statistically significant difference, at one or more concentrations. Concentration-related increases in the frequency of micronucleated cells and comparison to the vehicle control historical data will also be taken into account.
When inconclusive results are observed, or in case of toxic items when the highest analyzable dose-level does not exhibit about 50% toxicity, additional confirmatory experiments may be needed.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
ambiguous without metabolic activation
positive with metabolic activation

The test item induced chromosome damage, or damage to the cell division apparatus, in cultured L5178Y TK+/- mouse lymphoma cells, in the presence of a rat metabolizing system. In the absence of a rat metabolising system, its potential to induced chromosome damage, or damage to the cell division apparatus, remained equivocal.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells,in L5178Y TK+/-mouse lymphoma cells. The test was performed in accordance with OECD 487 guideline, and was GLP compliance.

 

Methods

After a preliminary toxicity test, the test item was tested in three independent experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

 

First experiment

Second experiment

Third experiment

Without S9 mix

3 h treatment +
24 h recovery

24 h treatment +
20 h recovery

 

With S9 mix

3 h treatment +
24 h recovery

3 h treatment +
24 h recovery

3 h treatment +
24 h recovery

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

The mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria, and positive controls showed clear unequivocal increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found severely cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

The dose-levels used for treatment were as follows:

.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL in the first experiment,

.           2.5, 5, 10, 15, 20, 25, 30 and 40 µg/mL in the second experiment.

 

Cytotoxicity

Following the 3-hour treatment, a marked to severe toxicity was observed at dose-levels = 37.5 µg/mL as shown by a 62 to 100% decrease in the PD.

Following the 24-hour treatment, a slight to marked toxicity was observed at 30 and 40 µg/mL as shown by a 32% and 72% decrease in the PD, respectively.


Micronucleus analysis

The dose-levels selected for micronucleus analysis were:

.           4.7, 9.4 and 18.8 µg/mL for the 3-hour treatment, higher dose-levels being too cytotoxic,

.           10, 20 and 30 µg/mL for the 24-hour treatment, higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of micronucleated cells was noted after the 3-hour treatment.

Following the 24-hour treatment, an increase in the frequency of micronucleated cells (exceeding the threshold of 2.5-fold the vehicle control value) was observed at 30 µg/mL. This increase was statistically significant (p < 0.05). However, the corresponding micronucleated cells remained within the historical data range of the vehicle control (5 micronucleated cells in 1000 cells versus 0-5 for the historical data). Consequently, this increase is considered as equivocal.

Experiments with S9 mix

The dose-levels used for treatment were as follows:

.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL for the first experiment,

.           4.7, 9.4, 18.8, 28.2, 37.5 and 75 µg/mL for the second experiment,

.           5, 10, 20, 30, 35, 40, 45 and 50 µg/mL for the third experiment.

 

Cytotoxicity

Following the first experiment, a moderate to severe toxicity was observed at dose-levels = 37.5 µg/mL as shown by a 45 to 100% decrease in the PD.

Following the second experiment, a slight to severe toxicity was noted at dose-levels = 9.4 µg/mL as shown by a 32% to 100% decrease in the PD.

Following the third experiment, a slight to severe toxicity was observed at dose-levels = 20 µg/mL as shown by a 25% to 100% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

.           9.4, 18.8 and 37.5 µg/mL for the first experiment, the latter inducing a 45% decrease in the PD,

.           4.7, 9.4 and 18.8 µg/mL for the second experiment, the latter inducing a 55% decrease in the PD,

.           10, 20 and 30 µg/mL for the third experiment, the latter inducing a 57% decrease in the PD.

 

In all experiments, slight increases in the frequency of micronucleated cells (exceeding the threshold of 2.5-fold the vehicle control value) were observed. These increases reached statistical significance in the first and third experiments and were dose-related. Therefore, these increases being reproducible in independent experiments, they were considered to be biologically significant.

 

Conclusion

The test item induced chromosome damage, or damage to the cell division apparatus, in cultured L5178Y TK+/-mouse lymphoma cells, in the presence of a rat metabolizing system. In the absence of a rat metabolising system, its potential to induced chromosome damage, or damage to the cell division apparatus, remained equivocal.

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:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, GLP, but no certificat of analysis was provided and the raw data are not presented. Some data are not reported, nevertheless the study is sufficiently detailed for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Procedure of Ames et al. (1975). Histidine requiring s. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100.The test was repeated twice: first with TA 1535 and TA 100, and subsequently again with TA 100.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay
Species / strain / cell type:
other: TA 1535, TA 153.7, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
a liver microsome fraction of Aroclor-induced rats
Test concentrations with justification for top dose:
0, 12, 37, 111, 333, 1000 µg/plate
Repeated test with only TA: 0, 11, 333, 1000, 3000, 6000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
other:
Positive control substance:
other: Sodium Azide for TA 1535 and TA 100 without S9; hypocanthone methanesulphonate for TA 1537, 1538 and TA98 without S9; 2-amino-anthracene for all strains in the presence of S9
Species / strain:
other: S. typhimurium TA 1535, 1537, 1538, 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
weak positive
Cytotoxicity / choice of top concentrations:
other: cytotoxicity hat highest concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

toxicity test:

test product per 0.1 ml per  plate background growth number of revertant colonies
100 o l) 0
10 0 0
1 SLD2 ) 60
0.1 NG3) 30
0.01 NG 53
0,00 NG 46

l) 0 = no growth. 2 ) SLD=  background lawn of  bacterial growth slightly less dense than in concomitant control plates.

3) NG  =normal background lawn  of  bacterial growth. Test with TA 100 (the results for the other strains are not in the attached file; clearly negative)
  STRAIN TA 100
  test material (µg/pl) Number of His + revertants (mean of 3 plates) SD
without S9 0 159 18
12 147 14
37 160 22
111 174 12
333 178 8
1000 257 17
       
With S9 0 163 17
12 123 12
37 156 15
111 180 13
333 206 15
1000 343 30

 

Repeat experiment with TA 100

  STRAIN TA 100
  test material (µg/pl) Number of His + revertants (mean of 3 plates) SD
without S9 0 145 (count of one plate, the other plates had to be excluded because of contamination)  
111 171 7
333 175 39
1000 293 26
3000 0 0
6000 0 0
       
With S9 0 157 33
111 145 17
333 193 15
1000 319 46
3000 0 0
6000 0 0

0 : no bacterial growth

1) count of one plate, the other plates had to be excluded because of a contamination

2 ) reference mutagens were tested in duplicate

Conclusions:
Interpretation of results (migrated information):
other: weakly positive only with TA 100, negative for all the other strains.

From the present results it is concluded that tert. amylhydroperoxide did not show any mutagen activity with strains TA 1535, TA 1537, TA
1538 or TA 98 either in the presence or in the absence of the liver microsome activation system under the test conditions employed in this evaluation.The significance of the slight increase in numbers of revertants with TA 100 was not clear, because at highest concentrations, the toxicity of the test liquid for the bacteria interfered with the mutagenicity testing.
Executive summary:

The potential of tert-amyl hydroperoxide to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 1538 TA 98, TA 100 ) was evaluated according to the Ames test (direct plate incorporation assay) and in compliance with GLP.

 Bacterias were exposed to the substance at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test : 0. 12, 37, 111, 333, 1000 µg/plate. After 72 hours of incubation at 37°C, the revertant colonies were scored. A second test was performed for TA 100 with the following concentrations: 0, 111, 333, 1000, 3000, 6000 µg/plate.

No noteworthy increase in the number of revertants was observed for all doses with and without metabolic activation on TA 1535, 1537, 1538 and 98. The significance of the slight increase in numbers of revertants with TA 100 was not clear, because at highest concentrations the toxicity of the test liquid for the bacteria interfered with the mutagenicity testing.

 

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:
1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable guideline study with acceptable restrictions: no certificat of analysis (but purity was provided), some details are missing
Qualifier:
no guideline followed
Principles of method if other than guideline:
The method is based on the "subculture method" of O'Neill and Hsie.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The test was carried out with Chinese hamster ovary (CHO) cells (subline CHO-K1, ATCC no CCL 61) maintained in Ham's F10 medium with newborn calf serum (15 %). The cells were originally obtained from Flow Laboratories, Irvine, Scotland.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (S9), derived from rats treated with Aroclor 1254 (500 mg/kg bodyweight)
Test concentrations with justification for top dose:
Without metabolic activation system: 0; 15; 40; 50 nl/ml
With metabolic activation system: 0; 40; 80; 100 nl/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methylsulphonate (EMS, 0.4 1/ml, without metabolic activation) and dimethylnitrosamine (DMN, 8 1/ml, with metabolic activation) were used as positive controls
Details on test system and experimental conditions:
The cellsare grown in Ham's F10 medium, supplemented with newborn calf serum (15 %), glutamine (2mM), streptomycin (50 mg/1) and penicillin G (50 mg/1). The cells are routinely passaged, twice weekly, after trypsinization. The split ratio is usually 1:10 for a confluent culture.
Evaluation criteria:
The mean number of initial and final survivors and mutants is calculated. The mutation frequency, that is to say the number of mutants per 100,000 clonable cells, is then calculated from the number of mutants and final survivors.
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of the toxicity test with tertiary amyl hydroperoxide with and without metabolic activation:

Test substnace (nl/ml, nominal) clonable cells (mean of three plates) survival (re. %)
Without metabolic activation
Control 358 100
0 306 85
5 315 88
10 227 63
20 152 42
40 23 6
60 12 3
80 5 1
With metabolic activation
Control 303 100
0 317 105
5 352 116
10 341 113
20 354 117
40 339 112
60 2 1
80 3 1

Results of the CHO/HPGRT test:

Test substance (nl/ml, nominal) Mean initial survival (rel. %) Mean final survival (%) Mutation frequency (per 100 000 clonable cells)
without metabolic activation
0 100 80 4.3
15 88 86 2.7
40 60 82 3.3
50 46 90 3.8
EMS (0,4 µl/ml) 80 81 30.1
with metabolic activation
0 100 80 1.8
40 95 96 4.2
80 88 82 2.4
100 80 90 4.2
DMN (8 µl/ml) <1 81 336.5
Conclusions:
Interpretation of results (migrated information):
negative

It can be concluded from the results of this study that tertiary amyl hydroperoxide does not induce mutations at the HGPRT locus in Chinese hamster ovary cells under the conditions of the test.
Executive summary:

Tert-amyl-hydroperoxide was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to the "subculture method" of O'Neill and Hsie.and in compliance with the Principles of Good Laboratory Practice. In a mammalian cell gene mutation assay (HPRT+/-), Chinese hamster ovary (CHO) cells (subline CHO-K1, ATCC no CCL 61) cells cultured were exposed in triplicate to the substance (purity 85) in DMSO for 5 hours at concentrations of 0, 15, 40, 50 nl/ml in the absence of metabolic activation and at concentrations of 0, 40, 80, 100 nl/ml in the presence of metabolic activation. Appropriate positive controls were used and showed a statistical increase in mutant colonies. Under these experimental conditions, tert-amyl-hydroperoxide did not induce any increase in mutant colonies and is not considered as mutagenic.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The genotoxic potential of tert-amyl hydroperoxide was investigated in the in vivo mammalian alkaline comet assay (Simar, 2016) performed under alkaline conditions, i. e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in liver, glandular stomach and forestomach of male rats in compliance with OECD Guideline 489 (2014) using 2 successive daily treatments at the maximum dose compatible with the toxicity of the test item, i. e. 200 mg/kg/day (x2), followed by one sampling time 2 to 6 hours after the last treatment. The two lower doses of 100 and 50 mg/kg/day (x2) were also analysed.

No statistically significant increases in the median percentage of DNA in tail of the hepatocytes, glandular stomach and forestomach cells were observed at the three analysed doses of 200, 100 and 50 mg/kg/day (x2), vs. the negative control.

Indeed, the means of median for the percentage of DNA in tail were 1.20, 1.57 and 3.10 % at 200, 100 and 50 mg/kg/day (x2) vs. 1.70% for the vehicle control group in hepatocytes, 30.50, 21.59 and 25.69 % vs. 22.49% in glandular stomach cells and 7.03, 11.47 and 13.26 % vs. 8.44% in forestomach cells, respectively.

Therefore, tert-amyl hydroperoxide is considered having no genotoxic activity in these organs.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 5 to 6 weeks old
- Weight at study initiation: between 165 g and 194 g (male)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: polypropylene cages
- Diet (ad libitum): A04C-10 from SAFE
- Water (ad libitum): softened by reverse osmosis and filtered on 0.22 µm membrane
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 50, 32, 20, 10 and 5 mg/mL
- Amount of vehicle (if gavage or dermal): 10ml/kg
Details on exposure:
The stability of the test item in the solvent was demonstrated for 9 days in previous repeated dose toxicity studies. Preparations for treatment were performed just before use.
Duration of treatment / exposure:
2 days
Frequency of treatment:
daily
Post exposure period:
3 to 6 hours after the last treatment.
Remarks:
Doses / Concentrations:
200, 320 and 500 mg/kg
Basis:
other: Toxicity assays
Remarks:
Doses / Concentrations:
50, 100 and 200 mg/kg
Basis:
other: Genotoxicity assay
No. of animals per sex per dose:
5-7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Methylmethane sulfonate (MMS) at a dose of 100 mg/kg PO, 24 hours and 3 to 6 hours before sacrifice.
Tissues and cell types examined:
Hepatocytes and glandular stomach and forestomach cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The OECD guideline recommends that the test be carried out at the maximum tolerated dose. The maximum tolerated dose is described as the highest dose which causes no mortality, but which may give rise to the appearance of signs of toxicity (OECD No. 489, 2014; Hayashi et al, 1994).
In absence of data regarding differences in systemic toxicity, metabolism, bioavailability, bone marrow toxicity, etc. including e.g. in a range-finding study, the preliminary toxicity test was performed in both sexes.
In an effort to reduce the use of laboratory animals required and to obtain more accurate information, the range- finding toxicity test was performed according to the improved experimental design recommended by Fielder et al. (1992). For the preliminary assay, groups of 2 male and 2 female rats 5 to 6 weeks old were treated. For the confirmatory assay, 10 animals were used (5 male and 5 female rats 5 to 6 weeks old).

DETAILS OF SLIDE PREPARATION:
The 5 males of each group were assigned for cell isolation and assessed for DNA fragmentation. Individual animals were anaesthetized with isoflurane and exsanguined. Single cell preparations were done within one hour after animal sacrifice. A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles was removed to reveal the abdominal cavity.
The whole liver, and the whole stomach were removed. The stomach and a portion of the liver that was previously cut were washed in the cold mincing buffer until as much blood as possible has been removed.

Isolation of hepatocytes
The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

Isolation of glandular stomach and forestomach cells
A portion of the stomach (containing both glandular stomach and forestomach) was removed as provision for histopathology (see § 10). The remaining stomach was separated in glandular stomach and forestomah to proceed to the comet assay on each specialised part of the organ.
The portions were minced with to release the cells. The cell suspensions were stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.
It is noteworthy that the whole stomachs were compacted when compared to the control organs. Moreover, at the highest dose of 200 mg/kg, the alimentary bolus was semiliquid, particularly at the forestomach level.

Dried slides preparation (pre-layering)
Conventional slides were dipped in hot 1.5 % Normal Melting Point Agarose in PBS. After gentle removal, the underside of the slides were wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry.

Slide preparation
Before use, a volume of 85 µL of 0.8% of Normal Agarose (NA) was added on the microscope slide pre-layered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2-8°C until the agarose layer hardens (3 to 5 minutes). The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75µL/slide) kept at ca. 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2-8°C.
Four slides per animal were prepared for the Comet assay (3 slides were actually read).

METHOD OF ANALYSIS:
Protocol for the Comet assay
Lysis
After the top layer of agarose has solidified, the glass coverslips were removed and the slides were immersed overnight at ca. + 4 °C in the dark in a lysing solution.

Unwinding, electrophoresis and staining
After this incubation period, the slides were then removed and placed on a horizontal gel electrophoresis unit and the unit filled with freshly prepared alkaline buffer to around 0.25 cm above the slides. In order to avoid excessive variation across the groups during each electrophoretic run, only one of the replicate slides were processed in each run for each animal (DNA – unwinding and electrophoresis). The cells were exposed to the alkali for ca. 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for ca. 20 minutes at <10°C by applying an electric current of 0.7 V / cm (25 V / 300 mA). All these steps were conducted protected from daylight to prevent the occurrence of additional DNA damage. After electrophoresis at pH >13, the slides were neutralized twice for ca. 5 minutes with 0.4 M Tris (pH 7.5) and the DNA was exposed for ca. 5 minutes to absolute ethanol in order to preserve all the Comet assay slides. Subsequently, the slides were air-dried and then stored at room temperature until they were scored for DNA migration.

Image analysis
Just prior to scoring, the DNA was stained using propidium iodide (final concentration of 20 µg/mL sterile water; 25 µL/slide).

Slides were examined with a 200 x magnification, using a fluorescent microscope (Leica Microsystems SAS - DM 2000, Heerbrugg, Switzerland), equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies), to the Comet Assay IV Image Analysis System, version 4.11 with Windows XP Pro Software (Perceptive Instruments Ltd, Suffolk, UK).

For all groups three slides were analysed with 50 nuclei per slide randomly scored. Five animals were retained per group, i.e. at least 15 slides per group, at least 750 analysed nuclei per group.

Tail parameters
Recent publications focused on the interpretation of the results through the analysis of the median of the percentage of DNA in tail, with the animal as statistical unit (D. Lovell and T. Omori, 2008).
In fact, this parameter appears to be the most linearly related to dose (B. Burlinson et al., 2007).

OTHER:
Cytotoxicity was evaluated through the enumeration of hedgehogs.
Evaluation criteria:
For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the concurrent negative control,
- This increase is dose-related when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage in the tissues studied in this test system.

A test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data for a given species, vehicle, route, tissue, and number of administrations
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.
Statistics:
In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
PRELIMINARY TOXICITY ASSAY
500 mg/kg/day (x1)
The preliminary assay performed on 2 male and 2 female rats at the dose of 500 mg/ kg/ day (x1) per os demonstrated 15 minutes after the treatment, in all animals a slight decrease in spontaneous motor activity, mydriasis and slight loss of muscular tonus in males, 25 minutes after the treatment.
20 to 30 minutes after the treatment, the females demonstrated titubation. Moreover, all animals presented 45 to 60 minutes after the treatment a moderate decrease in spontaneous motor activity, titubation, and a moderate loss of muscular tonus. Finally, 75 minutes after the first treatment, piloerection; was visible in all animals and the females demonstrated ptosis.
Under these conditions, for ethical reason, animals were sacrificed and the lower doses of 320 and 200 mg/kg were tested.

320 mg/kg/day (x2)
The dose of 320 mg/kg induced 15 min after the 1st treatment in all animals a slight increase in spontaneous, motor activity, moderate sniffing, and coprophagy. One hour after the 1st treatment, 1 male and 1 female demonstrated a moderate decrease in spontaneous motor activity, and the other male and female rats presented a moderate decrease in muscular tonus, slight tremors and a moderate chewing.
Moreover, 15 min after the 2nd treatment, the test item induced in all animals a moderate decrease in spontaneous motor activity and chewing, and 2 to 4 hours after the 2nd treatment all animals demonstrated a moderate decrease in spontaneous motor activity with slight decrease in muscular tonus.

200 mg/kg/day (x2)
The dose of 200 mg/kg induced 15 minutes after each treatment a slight decrease in spontaneous motor activity in all animals.

Under these conditions, the dose of 200 mg/kg was retained to be tested in the confirmatory toxicity assay.

CONFIRMATORY TOXICITY ASSAY
The dose of 200 mg/kg induced 15 minutes after each treatment a slight decrease in spontaneous motor activity in all animals.

The highest dose retained for the comet assay was set at 200 mg/kg/day (x2). Two inferior doses of 100 and 50 mg/kg/day (x2) were also tested.

As no sex-specific toxicity difference was observed between sexes in the preliminary toxicity assays, the comet test was performed using groups of 5 males only (7 males for the high dose group) treated at different dose levels. Indeed, as there was no substantial difference between sexes in toxicity, testing in a single sex is sufficient (OECD No. 489, 2014).

RESULTS OF DEFINITIVE STUDY
No statistically significant increases in the median percentage of DNA in tail of the hepatocytes at the three analysed doses of 200, 100 and 50 mg/kg/day (x2),vs.the negative control. Indeed, the means of median for the percentage of DNA in tail were of 1.20, 1.57 and 3.10 % at 200, 100 and 50 mg/kg/day (x2)vs. 1.70% for the vehicle control group

No statistically significant increases in the median percentage of DNA in tail of the glandular stomach cells at the three analysed doses of 200, 100 and 50 mg/kg/day (x2),vs.the negative control. Indeed, the means of median for the percentage of DNA in tail were of 30.50, 21.59 and 25.69 % at 200, 100 and 50 mg/kg/day (x2)vs. 22.49% for the vehicle control group.

No statistically significant increases in the median percentage of DNA in tail of the forestomach cells at the three analysed doses of 200, 100 and 50 mg/kg/day (x2),vs.the negative control. Indeed, the means of median for the percentage of DNA in tail were of 7.03, 11.47 and 13.26 % at 200, 100 and 50 mg/kg/day (x2) vs. 8.44% for the vehicle control group.

ORGAN: LIVER

 

in vivo COMET ASSAY IN ISOLATED RAT LIVER CELLS

 

 

 

GROUPS

 

 

 

TEST ITEM

 

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis1

 

p Mann- Whitney2

 

Relative ratio of hedgehogs3

 

p

Negative control

 

sterile water

0

1.70

 

 

 

 

N.S.

-

-

-

 

 

TREATED

 

 

tert-amyl hydroperoxide

200

1.20

N.S.

2.42

<0.01

100

1.57

N.S.

2.31

<0.01

50

3.10

N.S.

1.51

N.S.

Positive control

Methylmethane Sulfonate

 

100 mg/kg/day (x2)

53.23

-

<0.01

1.91

<0.05

ORGAN: GLANDULAR STOMACH

 

in vivo COMET ASSAY IN ISOLATED RAT GLANDULAR STOMACH CELLS

 

 

 

GROUPS

 

 

 

TEST ITEM

 

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis1

 

p Mann- Whitney2

 

Relative ratio of hedgehogs3

 

p

Negative control

 

sterile water

0

22.49

 

 

 

 

N.S.

-

-

-

 

 

TREATED

 

 

tert-amyl hydroperoxide

200

30.50

N.S.

0.56

<0.01

100

21.59

N.S.

0.82

N.S.

50

25.29

N.S.

0.78

N.S.

Positive control

Methylmethane Sulfonate

 

100 mg/kg/day (x2)

71.08

-

<0.01

0.99

N.S.

ORGAN: FORESTOMACH

 

in vivo COMET ASSAY IN ISOLATED RAT FORESTOMACH CELLS

 

 

 

GROUPS

 

 

 

TEST ITEM

 

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis1

 

p Mann- Whitney2

 

Relative ratio of hedgehogs3

 

p

Negative control

 

sterile water

0

8.68

 

 

 

 

N.S.

-

-

-

 

 

TREATED

 

 

tert-amyl hydroperoxide

200

7.03

N.S.

0.60

<0.01

100

11.47

N.S.

1.00

N.S.

50

13.26

N.S.

1.42

<0.05

Positive control

Methylmethane Sulfonate

 

100 mg/kg/day (x2)

48.83

-

<0.01

0.80

N.S.

1: Total group without positive control

2: Mean values obtained in treated group compared to Mean values obtained in solvent control group

3: Corresponds to the percentage of hedgehogs per treated group / percentage of hedgehogs in negative control group.

Conclusions:
Interpretation of results (migrated information): negative
Under these experimental conditions, tert-amyl hydroperoxide induced no statistically or biologically significant increases in DNA strand breaks at 200, 100 and 50mg/kg/day(x2) in male rat isolated Liver, Glandular Stomach and Forestomach cells after oral administration. Therefore, tert-amyl hydroperoxide is considered having no genotoxic activity in these organs.
Executive summary:

The genotoxic potential of tert-amyl hydroperoxide was investigated in the in vivo mammalian alkaline comet assay performed under alkaline conditions, i.e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in liver, glandular stomach and forestomach of male rats in compliance with OECD Guideline 489 (2014) using 2 successive daily treatments at the maximum dose compatible with the toxicity of the test item, i.e. 200 mg/kg/day (x2), followed by one sampling time 2 to 6 hours after the last treatment. The two lower doses of 100 and 50 mg/kg/day (x2) were also analysed.

No statistically significant increases in the median percentage of DNA in tail of the hepatocytes, glandular stomach and forestomach cells were observed at the three analysed doses of 200, 100 and 50 mg/kg/day (x2), vs. the negative control.

Indeed, the means of median for the percentage of DNA in tail were 1.20, 1.57 and 3.10 % at 200, 100 and 50 mg/kg/day (x2) vs. 1.70% for the vehicle control group in hepatocytes, 30.50, 21.59 and 25.69 % vs. 22.49% in glandular stomach cells and 7.03, 11.47 and 13.26 % vs. 8.44% in forestomach cells, respectively.

Therefore, tert-amyl hydroperoxide is considered having no genotoxic activity in these organs.

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

Additional information

Justification for classification or non-classification

Three in vitro and one in vivo tests are available and allow to conclude that tert-amyl hydroperoxide in not genotoxic. Ames test (TNO, 1981): inconclusive on TA100. HPRT (TNO, 1982): negative. In vitro micronucleus test (Sarlang, 2012): positive. Comet assay on liver and gladular and forestomach cells (Simar, 2016): negative.

According to EU Regulation (EC) N0. 1272/2008 (CLP), the substance is not classified for genotoxicity.